HK40113869A - Aptamers for personal health care applications - Google Patents

Description

aptamers for personal healthcare applications

Technical Field

This article describes a nucleic acid aptamer with high binding affinity and specificity to cell membrane glycoproteins, and preferably to intercellular adhesion molecule-1 (“ICAM-1”), and more specifically, describes the use of such aptamers to inhibit human rhinovirus from binding to this glycoprotein and entering cells in the nasal cavity and pharynx.

The sequence list is incorporated by reference.

This application contains a sequence list in computer-readable form (filename: CM05385M.txt; size: 337 kilobytes; creation date: December 1, 2022) which is a separate part of this disclosure and is incorporated herein by reference in its entirety.

Background Technology

Aptamers are short, single-chain oligonucleotides with specific and complex three-dimensional shapes that bind to target molecules. Aptamer molecular recognition is based on structural compatibility and intermolecular interactions, including electrostatic forces, van der Waals interactions, hydrogen bonds, and π-π stacking interactions between the aromatic ring and the target material. Aptamer targets include, but are not limited to, peptides, proteins, nucleotides, amino acids, antibiotics, low-molecular-weight organic or inorganic compounds, and even whole cells. The dissociation constant of aptamers typically varies between the micromolar and picomolar levels, comparable to the affinity of antibodies for their antigens. Aptamers can also be engineered to exhibit high specificity, enabling them to distinguish target molecules from closely related derivatives.

Aptamers are typically designed in vitro from large random nucleic acid libraries using Systematic Evolution of Ligands with Exponentially Enriched Ligands (SELEX). The SELEX method was first introduced in 1990 when selecting single-stranded RNA for low molecular weight dyes (Ellington, A.D., Szostak, J.W., 1990. Nature 346:818-822). A few years later, single-stranded DNA aptamers and aptamers containing chemically modified nucleotides were also described (Ellington, A.D., Szostak, J.W., 1992. Nature 355:850-852; Green, L.S., et al., 1995. Chem. Biol. Vol. 2: 683–695). Since then, aptamers have been selected for hundreds of microscopic targets such as cations, small molecules, proteins, cells, or tissues. A compilation of examples from the literature is available in the database at: http://www.aptagen.com/aptamer-index/aptamer-list.aspx.

The common cold is the most prevalent illness in the United States, infecting 62 million people annually. Adults typically catch the cold 2-4 times a year, while children catch it 8-12 times. This leads to illness, frequent absences, decreased productivity, and inappropriate antibiotic use. This translates to $60 billion in costs for the United States each year.

Human rhinoviruses cause 50%–80% of colds. Rhinoviruses are small (30 nm), non-enveloped, single-stranded RNA viruses. Although rhinovirus infection is mild and self-limiting in immune-active hosts, it is associated with pneumonia in immunocompromised patients, bronchiolitis in infants and young children, and can exacerbate pre-existing lung conditions such as asthma and chronic obstructive pulmonary disease.

Rhinovirus infection primarily occurs in the nasopharynx when the virus attaches to surface receptors on the nasal epithelium and infects host cells. Fifty-seven percent of rhinoviruses attach to the ICAM-1 receptor along the airway. Once inside a cell, the virus hijacks the cell's replication mechanisms to make copies of itself. This leads to cell lysis and death, allowing viral progeny to spread to other nearby cells to repeat the infection cycle. Ultimately, this triggers a host immune response, resulting in respiratory symptoms (e.g., cough, runny nose, nasal congestion, sore throat, etc.). Despite the heavy public health burden, there are no licensed vaccines or antiviral drugs for human rhinovirus.

Aptamers targeting proteins such as intercellular adhesion molecule 1 (ICAM-1) have been previously described. However, no data have been reported on the binding of these aptamers to membrane-bound proteins or their ability to prevent the binding of natural ligands or human rhinovirus to ICAM-1. Therefore, there remains a need for aptamers that selectively bind to cell membrane glycoproteins (including ICAM-1) and prevent human rhinovirus from binding to these glycoproteins, thereby alleviating cold symptoms or preventing (re)infection.

Summary of the Invention

This article describes the use of SELEX for selecting parental aptamers for intercellular adhesion molecule 1 (ICAM-1) and illustrates the use of aptamers with secondary structures defined herein for preventing human rhinovirus from binding to this glycoprotein. The aptamers can be truncated from the parental sequence selected by SELEX.

This document also describes an aptamer composition. The aptamer composition comprises at least one oligonucleotide, the at least one oligonucleotide comprising: deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof; wherein the aptamer composition has binding affinity for intercellular adhesion molecule 1 (ICAM-1), and wherein the aptamer composition is configured to reduce the binding of one or more human rhinoviruses to said intercellular adhesion molecule 1 (ICAM-1), and wherein the aptamer composition comprises at least one secondary structure forming a secondary structure from the 5′-end to the 3′-end. An oligonucleotide, the secondary structure comprising at least a 5′-overhanging end, a stem, a hairpin loop, and a 3′-overhanging end, wherein the stem is formed between the hairpin loop and the overhanging end; and wherein the secondary structure comprises at least the structural sequence 5′-EEEYYSSSSYSSSSYHHHHYYYYYYYSYYYYYEEEE-3′ (structure I), wherein H represents a nucleotide located in the hairpin loop, S represents a nucleotide located in the stem, E represents a nucleotide located in the overhanging end, and Y represents the absence of the nucleotide or H, S, E, or I, wherein I represents an inner loop.

Aptamer compositions may further exhibit binding affinity for one or more of the following: members of the low-density lipoprotein receptor (LDLR) family, cadherin-associated family member 3 (CDHR3), and combinations thereof.

The aptamer composition may comprise at least one oligonucleotide exhibiting at least 80% nucleotide sequence identity with an oligonucleotide selected from the group consisting of: SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, and SEQ ID NO:246.

This document also describes a personal healthcare composition. The personal healthcare composition comprises an aptamer composition as described herein. The personal healthcare composition may comprise at least one nucleic acid aptamer; wherein the nucleic acid aptamer has binding affinity for intercellular adhesion molecule 1 (ICAM-1), wherein the nucleic acid aptamer is configured to reduce the binding of one or more human rhinoviruses to ICAM-1, and wherein the aptamer composition comprises at least one oligonucleotide forming a secondary structure from a 5′-end to a 3′-end, the secondary structure comprising at least a 5′-overhanging end, a stem, a hairpin loop, and a 3′-overhanging end, wherein the stem is formed between the hairpin loop and the overhanging ends;

The secondary structure includes at least the structural sequence 5′-EEEYYSSSSYSSSSYHHHHYYYYYYYYSYYYYYEEEE-3′ (structure I), where H represents a nucleotide in the hairpin loop, S represents a nucleotide in the stem, E represents a nucleotide in the dangling end, and Y represents the absence of a nucleotide or H, S, E, or I, where I represents an inner loop.

In one aspect, the healthcare composition may contain at least one oligonucleotide that exhibits at least 80% nucleotide sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246.

This document also describes an aptamer composition comprising at least one peptide or protein, wherein the peptide or protein is translated from at least one truncated oligonucleotide disclosed herein.

This personal healthcare composition may also exhibit binding affinity for one or more of the following: members of the low-density lipoprotein receptor (LDLR) family, cadherin-associated family member 3 (CDHR3), and combinations thereof.

A method for delivering a personal healthcare composition disclosed herein to the upper respiratory tract is also provided. The method includes administering the personal healthcare composition as described herein; the personal healthcare composition comprising at least one nucleic acid aptamer; wherein the at least one nucleic acid aptamer has binding affinity for intercellular adhesion molecule 1 (ICAM-1), wherein the nucleic acid aptamer is configured to reduce the binding of one or more human rhinoviruses to ICAM-1, and wherein the aptamer composition comprises at least one oligonucleotide forming a secondary structure from a 5′-end to a 3′-end, the secondary structure comprising at least a 5′-overhanging end, a stem, a hairpin loop, and a 3′-overhanging end, wherein the stem is formed between the hairpin loop and the overhanging ends; and

The secondary structure includes at least the structural sequence 5′-EEEYYSSSSYSSSSYHHHHYYYYYYYYSYYYYYEEEE-3′ (structure I), where H represents a nucleotide in the hairpin loop, S represents a nucleotide in the stem, E represents a nucleotide in the dangling end, and Y represents the absence of a nucleotide or H, S, E, or I, where I represents an inner loop.

In one aspect, the healthcare composition may contain at least one oligonucleotide that exhibits at least 80% nucleotide sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246.

In one aspect, the personal healthcare composition may also contain one or more additional active ingredients; wherein at least one nucleic acid aptamer and one or more active ingredients are covalently or non-covalently attached.

This document also describes the use of the aptamer compositions disclosed herein and/or the personal healthcare compositions disclosed herein for the purpose of inhibiting human rhinovirus infection by inhibiting binding to intercellular adhesion molecule 1 (ICAM-1) and thereby inhibiting viral entry into cells within the nasal cavity and pharynx. This use may include delivery of the aptamer compositions and/or personal healthcare compositions disclosed herein to the upper respiratory tract. In one aspect, at least one oligonucleotide showing at least 80% nucleotide sequence identity with an oligonucleotide sequence selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246 may be used.

Attached Figure Description

Although this specification concludes by specifically pointing out and clearly claiming the subject matter of the invention, it is believed that the invention will be more readily understood from the following description taken in conjunction with the accompanying drawings, wherein:

Figure 1 shows a schematic diagram of the DNA library.

Figure 2 illustrates the parent aptamer selection strategy during selection rounds 1 through 11.

Figure 3 illustrates the parental aptamer splitting selection strategy during selection rounds 12 to 14.

Figure 4 shows the enrichment trajectories of the first twenty parental aptamers.

Figure 5 shows the binding assay results of the selected parental aptamers on human nasal epithelial cells (HNEpC) and HEK293 cells.

Figures 6A to 6D show the fluorescently labeled parental aptamer Nas.R-4 (SEQ ID NO: 04) that binds to HNEpC cells and HEK293 cells, wherein Figure 6A shows a fluorescence image, Figure 6B shows a bright-field image of HNEp cells, Figure 6C shows a fluorescence image, and Figure 6D shows a bright-field image of HEK293 cells.

Figures 7A through 7H illustrate the virus inhibition assays on HeLa cells using the parental aptamer Nas.R-2 (SEQ ID NO: 02), the parental aptamer Nas.R-8, and the negative control aptamer. Figure 7A shows a fluorescence image, and Figure 7B shows a bright-field image using the Nas.R-2 aptamer; Figure 7C shows a fluorescence image, and Figure 7D shows a bright-field image using the Nas.R-8 aptamer; Figure 7E shows a fluorescence image, and Figure 7F shows a bright-field image using the control aptamer; Figure 7G shows a fluorescence image, and Figure 7H shows only a bright-field image of the cells.

Figure 8 shows the binding affinity of the parental aptamers Nas.R-1, Nas.R-2, Nas.R-4, and Nas.R-8 (SEQ ID NO: 01, 02, 04, and 08) to 250 nM exogenous ICAM-1, determined by surface plasmon resonance (SPR).

Figure 9 shows the binding affinity of the parental aptamers Nas.R-1, Nas.R-2, Nas.R-4, and Nas.R-8 (SEQ ID NO: 01, 02, 04, and 08) to 250 nM human serum albumin as a control, determined by surface plasmon resonance (SPR).

Figure 10 shows the amino acid sequence alignment of the extracellular domains of ICAM-1, ICAM-3, and ICAM-5 (SEQ ID NO: 214, 232, and 263).

Figure 11 shows examples of sequences that exhibited higher enrichment levels in nasal cell positive selection than in HEK293 cell positive selection. Data points are presented after 12, 13, and 14 selection rounds.

Figure 12 shows an example of a sequence from selection round 14, which exhibited a higher enrichment level in HEK293 positive selection than in positive selection targeting nasal cells. Data points are presented after selection rounds 12, 13, and 14.

Figure 13 shows an alignment of exemplary sequences identified during the selection process that have at least 90% nucleotide sequence identity.

Figure 14 shows the alignment of exemplary sequences with at least 70% nucleotide sequence identity identified during the selection process.

Figure 15 shows the alignment of exemplary sequences with at least 50% nucleotide sequence identity identified during the selection process.

Figure 16 shows the results of motif analysis of random regions of the parent aptamer Nas.D-1 (SEQ ID NO:101).

Figure 17 shows the parental aptamer Nas.R-1 (SEQ ID NO: 01) and its predicted secondary structure of the conserved motif highlighted in circles.

Figure 18 shows a motif analysis based on the motif frequencies of random regions shown as the first 100 parental aptamers of the DNA sequence.

Figure 19 shows the results of motif analysis of random regions of aptamer Nas.D-4 (SEQ ID NO:104).

Figure 20A shows the predicted secondary structure of the parent aptamer Nas.D-4 (SEQ ID NO: 104). Figures 20B and 20C show two alternative predicted secondary structures of the aptamer truncated from Nas.D-4 (SEQ ID NO: 244) (shown by the dashed line in Figure 20A).

Figure 21 shows the results of motif analysis of random regions of the parent aptamer Nas.D-8 (SEQ ID NO:108).

Figure 22A shows the predicted secondary structure of the parental aptamer Nas.D-8 (SEQ ID NO: 108). Figure 22B shows the predicted secondary structure of the aptamer truncated from Nas.D-8 (SEQ ID NO: 246) (shown by the dashed line in Figure 22A), which includes the two mutations indicated by the dashed circles.

Figure 23 shows the binding affinity of the truncated aptamer of SEQ ID NO. 242 (denoted as 2.1) to the parental aptamer sequence SEQ ID NO. 102 (denoted as 2.0).

Figure 24 shows the binding affinity of the truncated aptamer of SEQ ID NO. 244 (denoted as 4.1) to the parental aptamer sequence SEQ ID NO. 104 (denoted as 4.0).

Figure 25 shows the binding affinity of the truncated aptamer of SEQ ID NO. 245 (denoted as 5.1) to the parental aptamer sequence SEQ ID NO. 105 (denoted as 5.0).

Figure 26 shows the binding affinity of the truncated aptamer of SEQ ID NO. 246 (denoted as 8.1) to the parental aptamer sequence SEQ ID NO. 108 (denoted as 8.0).

Figure 27A shows the first three structural motifs formed in the secondary structure of the aptamer disclosed herein. Figure 27B shows the distribution of the motifs shown in Figure 27A in the example sequence.

Figure 28 illustrates the efficacy of aptamers including special sequence motifs in binding experiments.

Figure 29 shows the most likely secondary structures of mutant aptamer 4.1 (denoted as state 1) of SEQ ID NO:264 at 33 °C and 37 °C.

Figure 30 shows the most likely secondary structures of mutant aptamer 4.1 (denoted as state 2) of SEQ ID NO:265 at 33 °C and 37 °C.

Figure 31 shows the three most likely secondary structures that the mutant aptamer state 2 will form; Figure 31A shows the most likely secondary structure (denoted as structure 1), Figure 31B shows the second most likely secondary structure (denoted as structure 2), and Figure 31C shows the third most likely secondary structure (denoted as structure 3).

Figure 32 shows the distribution of the secondary structure of mutant aptamer state 2; Figure 32A shows the distribution at 33°C and Figure 32B shows the distribution at 37°C.

Figure 33A shows the most likely secondary structure of mutant aptamer 4.1 (denoted as state 4) of SEQ ID NO:267 at 33 °C, and Figure 33B shows the most likely secondary structure of mutant aptamer 4.1 (denoted as state 4) of SEQ ID NO:267 at 37 °C.

Detailed Implementation

I. Definition

As used in this article, the term "aptamer" refers to a single-chain oligonucleotide or peptide that has a binding affinity for a specific target.

As used in this article, the term "nucleic acid" refers to a polymer or oligomer of nucleotides. When the sugar portion of a nucleotide is D-ribose, the nucleic acid is also called "ribonucleic acid" (RNA), and when the sugar portion is 2-deoxy-D-ribose, the nucleic acid is also called "deoxyribonucleic acid" (DNA).

As used herein, the term "nucleotide" refers to a compound consisting of a nucleoside esterified via a hydroxyl group at the 5-carbon of its sugar moiety into a monophosphate, polyphosphate, or phosphate-derived group. When the sugar moiety is D-ribose, the nucleotide is also referred to as a "ribonucleotide," and when the sugar moiety is 2-deoxy-D-ribose, the nucleotide is also referred to as a "deoxyribonucleotide." In the compositions, uses, methods, and applications disclosed herein, ribonucleotides may be replaced by deoxyribonucleotides, and vice versa.

As used herein, the term "nucleoside" refers to a glycoamine composed of a nucleobase, such as a purine or pyrimidine, typically linked by a β-glycosidic bond to a 5-carbon sugar (e.g., D-ribose or 2-deoxy-D-ribose). When the sugar moiety is D-ribose, the nucleoside is also referred to as a "ribonucleotide," and when the sugar moiety is 2-deoxy-D-ribose, the nucleoside is also referred to as a "deoxynucleotide."

As used herein, the term "nucleobase" refers to a nitrogen-containing compound that has the chemical properties of a base. Non-limiting examples of nucleobases include compounds containing pyridine, purine, or pyrimidine moieties, including but not limited to adenine, guanine, hypoxanthine, thymine, cytosine, and uracil.

As used in this article, the term "oligonucleotide" refers to oligomers composed of nucleotides.

As used herein, in the context of two or more oligonucleotides, nucleic acids, or aptamers, the term “identical” or “sequence identity” refers to sequences of two or more identical or having a specified percentage of identical nucleotides when compared and aligned to achieve maximum correspondence, such as when measured by sequence comparison algorithms or by visual inspection.

As used herein, in the context of two or more oligonucleotides, nucleic acids, or aptamers, the terms “substantially homologous” or “substantially identical” generally refer to two or more sequences or subsequences that have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide identity when compared and aligned to achieve maximum correspondence, whether measured by sequence comparison algorithms or by visual inspection.

As used herein, the term "epitope" refers to a region of the target that interacts with the aptamer. An epitope can be an adjacent segment within the target, or it can be represented by multiple physically adjacent points in the folded form of the target.

As used herein, the term "motif" refers to a continuous or sequence of continuous nucleotide sequences present in a library of aptamers with binding affinity to a specific target, and exhibiting a statistically significantly higher probability of occurrence than expected compared to a random oligonucleotide library. Motif sequences are often a result of or a driving factor in the aptamer selection process.

As used herein, the term "personal healthcare composition" refers to a composition in a form that can be delivered directly to the upper respiratory tract.

As used in this article, "pharmaceuticalally effective amount" means an amount sufficient to impart a therapeutic effect to a subject. In some respects, therapeutic effects reduce the binding of rhinovirus to cell membrane glycoproteins, such as ICAM-1, reduce the severity and/or duration of the common cold, or reduce the incidence of respiratory illness caused by rhinovirus.

As used herein, the term “parent” (preferably in combination with sequence, oligonucleotide and/or aptamer) will be used for molecules that are further modified, particularly shortened or truncated to shorter portions, preferably sequences, oligonucleotides and/or aptamers.

As used herein, the term “truncated” (preferably in combination with sequence, oligonucleotide and/or aptamer) will be used for molecules, preferably sequences, oligonucleotides and/or aptamers, truncated from parent molecules.

As used herein, the term "hybrid" refers to the Watson/Crick base pairing known in the art, in which a nucleotide pairs with a specific other nucleotide due to hydrogen bonding. Thus, adenine pairs with thymine, and cytosine pairs with guanine; alternatively, thymine may also pair with guanine. The term nucleotide pair will have the same meaning in this sense as "hybrid."

As used herein, the term "inner" refers to the structure separated from the two ends (5'-end and 3'-end) of the aptamer by nucleotides that hybridize with each other ("stem" as defined below). For consistency, the term "outer" refers to the structure adjacent to either end nucleotide (5'-end or 3'-end).

As used herein, the term "loop" formed in secondary structures refers to one or more unpaired inner nucleotides. Examples of "loops" in secondary structures are hairpin loops or inner loops.

As used herein, the term "hairpin loop" refers to a loop formed within a secondary structure, where the loop is formed only near a "stem" region. The letter "H" will be used in the structural sequence of the nucleotides that form the hairpin loop.

As used herein, the term "inner loop" refers to a loop formed within a secondary structure, where the loop is formed near two or more "stem" regions. The letter "I" will be used in the structural sequence of the nucleotides that form the inner loop.

As used herein, the term "stem" refers to a region containing paired nucleotides, wherein at least one nucleotide pair is formed, preferably two or three consecutive nucleotide pairs, to construct a stem. The letter "S" will be used in the structural sequence of the nucleotides forming the stem.

As used herein, the term "terminus" refers to a region within a secondary structure that includes at least one first nucleotide at the 5' end of an oligonucleotide or at least one last nucleotide at the 3' end of an oligonucleotide. The letter "E" will be used in the structural sequence of the nucleotides forming the terminus of an oligonucleotide.

As used herein, the term "dangling end" is used if at least one first nucleotide at the 5' end does not hybridize with another nucleotide, and/or at least one last nucleotide at the 3' end does not hybridize with another nucleotide. Thus, the corresponding end is referred to as "dangling" because the end remains empty only from one point.

II. Aptamer Composition

Human rhinoviruses (RVs) are the primary cause of the common cold. They are classified into three groups (RV-A, RV-B, and RV-C), encompassing approximately 160 types that express different surface proteins. Despite this diversity, rhinoviruses primarily utilize three glycoproteins from epithelial cells to cross the cell membrane and enter the host cell for replication: intercellular adhesion molecule 1 (ICAM-1) protein, used by most RV-A types and all RV-B types; low-density lipoprotein receptor (LDLR) or LDLR family members, used by at least twelve RV-A types; and cadherin-associated family member 3 (CADHR3) protein, primarily used by RV-C types.

The aptamer composition comprises at least one oligonucleotide selected from the group consisting of: deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof, wherein the aptamer composition has binding affinity for intercellular adhesion molecule 1 (ICAM-1). Deoxyribonucleotide-based and ribonucleotide-based aptamers are included in this invention. Deoxyribonucleotide-based aptamers may be advantageous in some examples due to their lower cost and higher oligonucleotide stability. In one aspect, the aptamer composition may have binding affinity for one or more cell membrane glycoproteins selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), members of the low-density lipoprotein receptor (LDLR) family, and cadherin-associated family member 3 (CDHR3), and combinations thereof. The aptamer composition is configured to reduce or inhibit the binding of one or more human rhinoviruses to intercellular adhesion molecule 1 (ICAM-1). The aptamer composition may comprise at least one oligonucleotide, wherein the at least one oligonucleotide comprises a secondary structure providing binding affinity for the ICAM-1 molecule. Suitable oligonucleotides forming the secondary structure comprise 30 to 60 nucleotides, or 30 to 55 nucleotides, or 30 to 45 nucleotides, or 35 to 40 nucleotides in length. The formed secondary structure may include at least one double-stranded stem region, at least one hairpin loop formed by unpaired/unhybridized nucleotides, and a 5′-end and a 3′-end, or a combination thereof. The 5′-end and 3′-end may be dangling ends not integrated into any other structural motif. An example loop formed by the secondary structures disclosed herein is a hairpin. A stable hairpin is formed if the bases at either end of adjacent unhybridized bases hybridize with each other. The secondary structures disclosed herein may also include a second unpaired region forming an inner loop comprising adjacent unhybridized bases, each side of which is a different hybridized base (stem). The size and length of the unpaired loop and the double-stranded stem can vary. Suitable sizes for example loops to be formed in the secondary structures of the sequences disclosed herein are in the range of 1 to 15 unpaired nucleotides, or 1 to 10 unpaired nucleotides, or 2 to 8 unpaired nucleotides. Example hairpin loops may include at least 3 or more unpaired nucleotides, or 4 or more unpaired nucleotides, or 5 or more unpaired nucleotides. Example inner loops may include at least 1 unpaired nucleotide, or 1 to 5 unpaired nucleotides, or 1 to 10 unpaired nucleotides. Suitable sizes for example double-stranded stems to be formed in the secondary structures of the sequences disclosed herein are in the range of 1 to 15 paired nucleotides, or 1 to 10 paired nucleotides, or 2 to 8 paired nucleotides, or 3 to 6 paired nucleotides, particularly for second stems located near the 5′-overhang and/or 3′-overhang ends.

The lengths of the 5′-overhang and/or 3′-overhang can also vary, but a suitable length for the overhang can include at least one unpaired/unhybridized nucleotide, or at least three unpaired/unhybridized nucleotides, or at least four unpaired/unhybridized nucleotides, or at least five unpaired/unhybridized nucleotides. For example, the 5′-overhang and 3′-overhang can extend to up to 15 unpaired/unhybridized nucleotides, but the secondary structure of the aptamer must be conserved. The suitable lengths of the overhangs in the secondary structures disclosed herein range from three to 15 unpaired/unhybridized nucleotides.

Suitable secondary structures demonstrating binding affinity for ICAM-1 and configured to inhibit binding to human rhinovirus may include the structural sequence from the 5′-end to the 3′-end: 5′-EEEYYSSSSYSSSSYHHHHYYYYYYYYSYYYYYEEEE-3′ (Structure I), where H represents the nucleotide forming the hairpin loop, S represents the nucleotide located in the stem, E represents the nucleotide located in the dangling end, and Y represents the absence of the nucleotide or H, S, E, or I, where I represents the inner loop as classified herein. This structural sequence is based on P. Danaee et al.; Nucleic Acids Research, Vol. 46, No. 11, June 20, 2018, pp. 5381-5394; this literature is incorporated herein by reference. The hairpin loop in Structure I may include at least one purine base, or the hairpin loop may include at least three pyrimidine bases, or the hairpin loop may include a combination thereof. The hairpin loop in Structure I has a length of at least four unpaired/unhybridized nucleotides, but suitable lengths of hairpin loops disclosed herein may include five or more unpaired/unhybridized nucleotides, wherein the hairpin may, for example, be formed such that the first nucleotide in the hairpin loop, starting from the 5′ end, is a purine base. To form the hairpin loop of unpaired/unhybridized nucleotides, a stem region comprising paired nucleotides and closing the hairpin loop is formed in Structure I.

In the suitable secondary structures disclosed herein and exemplarily illustrated in Structure I, the last nucleotide in the stem region preceding the hairpin loop starting at the 5′ end may be a purine base and/or the first nucleotide in the stem following the hairpin loop starting at the 5′ end may be a pyrimidine base. Alternatively, the first nucleotide in the stem adjacent to the 5′-overhang may be a pyrimidine base, wherein the stem region between the 5′-overhang and the hairpin loop may further include a three-nucleotide sequence motif, namely the motif thymine-cytosine-adenine (TCA).

In the suitable secondary structures disclosed herein and exemplarily illustrated in Structure I, suitable bases may also be present in the stem adjacent to the 3′-overhang, wherein the last nucleotide in the stem, starting from the 5′-end and adjacent to the 3′-overhang, may be a purine base. Alternatively or additionally, the stem adjacent to the 3′-overhang may also include a 3-nucleotide sequence motif, namely the motif guanine-cytosine-thymine (GCT). The length of the stem adjacent to the overhang may vary and may include at least 3 hybrid base pairs, at least 4 hybrid base pairs, or at least 5 hybrid base pairs.

The length of the dangling end can also vary, as exemplarily shown in Structure I. For example, the 5′-dangling end may include at least four unpaired nucleotides, in one example four to fifteen unpaired nucleotides, wherein at least two of the unpaired nucleotides may be purine bases. Alternatively or alternatively, the 3′-dangling end may include at least three or four unpaired nucleotides, in one example three to fifteen unpaired nucleotides, wherein at least four of the unpaired nucleotides in the 3′-dangling end are purine bases. Longer 3′-dangling ends advantageously include five, six, or seven unpaired nucleotides, all of which may be purine bases. Alternatively or alternatively, the 3′-dangling end may also include a sequence motif comprising four to seven unpaired nucleotides, namely the motif GAGGYYZ, wherein Y and Z are absent or selected from guanine (G), cytosine (C), adenosine (A), thymine (T), or uracil (U). In one aspect, Y can be A or G; or Y can be A in the GAGGYYZ motif. The GAGGYYZ motif can be at least partially unhybridized, and advantageously, the motif can be completely unhybridized. In one aspect, the GAGGYYZ motif can be located near and/or at the 3′ end. For example, Y in the GAGGYYZ motif can be at least one nucleotide from the last 15 nucleotides and/or Z in the GAGGYYZ motif can be at least one nucleotide from the last 13 nucleotides or the last nucleotide at the 3′ end. Thus, the 3′ end can be a dangling end, and the motif can be located entirely at the dangling end or still partially in the stem region. For example, GAG in the GAGGYYZ motif can be paired in the stem region and/or GYYZ in the GAGGYYZ motif can be unpaired at the 3′ dangling end. In one aspect, the entire GAGGYYZ motif can be unpaired at the 3′ dangling end.

At least one oligonucleotide forming the secondary structure disclosed herein can be truncated from the parental sequence disclosed herein. The aptamer composition may comprise at least one oligonucleotide comprising about 30 to about 60 consecutive nucleotides truncated from the group consisting of SEQ ID NO:1 to SEQ ID NO:200, which are parental sequences. The aptamer composition may comprise at least one oligonucleotide comprising at least 32 consecutive nucleotides truncated from the parental sequence disclosed herein. The aptamer composition may comprise at least one oligonucleotide comprising at least 33 consecutive nucleotides truncated from the parental sequence disclosed herein. The aptamer composition may comprise at least one oligonucleotide comprising at least 34 consecutive nucleotides truncated from the parental sequence disclosed herein. The aptamer composition may comprise at least one oligonucleotide comprising at least 35 consecutive nucleotides truncated from the parental sequence disclosed herein. The aptamer composition may comprise at least one oligonucleotide comprising at least 36 consecutive nucleotides truncated from the parental sequence disclosed herein. The aptamer composition may contain at least one oligonucleotide comprising at least 37 consecutive nucleotides truncated from the parental sequence disclosed herein.

Alternatively, the aptamer composition may include at least one oligonucleotide forming the secondary structure disclosed herein and truncated from the 3′ end, 5′ end, or middle of the parental sequence disclosed herein, for example selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200, which are parental sequences. The aptamer composition may comprise at least one oligonucleotide truncated from the middle of the parental sequence disclosed herein.

At least one truncated oligonucleotide of the present invention may begin or end at nucleotide 12, calculated from the 3′ end of the parental sequence disclosed herein. At least one truncated oligonucleotide of the present invention may begin or end at nucleotide 10, calculated from the 3′ end of the parental sequence disclosed herein. At least one truncated oligonucleotide of the present invention may begin or end at any nucleotide from the 3′ end to nucleotide 9, calculated from the 3′ end of the parental sequence disclosed herein.

The aptamer composition may comprise at least one oligonucleotide forming the secondary structure disclosed herein and truncated from a group consisting of oligonucleotides as parental sequences, the at least one oligonucleotide having at least 80% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200, wherein the truncated oligonucleotide contains the GAGGYYZ motif disclosed herein, including but not limited to all variants and additional specifications. The aptamer composition may comprise at least one oligonucleotide truncated from a group consisting of oligonucleotides as parental sequences, the at least one oligonucleotide having at least 90% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200. A non-limiting example of an oligonucleotide having at least 90% nucleotide sequence identity with SEQ ID NO:3 is, for example, SEQ ID NO:88. The aptamer composition may comprise at least one oligonucleotide, which is at least one oligonucleotide truncated from a group consisting of oligonucleotides serving as a parent sequence, and which has at least 93% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200. The aptamer composition may comprise at least one oligonucleotide, which is at least one oligonucleotide truncated from a group consisting of oligonucleotides serving as a parent sequence, and which has at least 96% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200. The aptamer composition may comprise at least one oligonucleotide, which is at least one oligonucleotide truncated from a group consisting of oligonucleotides serving as a parent sequence, and which has at least 99% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, the at least one oligonucleotide showing less than 100% nucleotide sequence identity, wherein a nucleotide having a pyrimidine base is replaced by another nucleotide having a pyrimidine base, a nucleotide having a purine base is replaced by another nucleotide having a purine base, or a combination thereof.

The aptamer composition may comprise at least one oligonucleotide forming the secondary structure disclosed herein and truncated from the group consisting of SEQ ID NO:1 to SEQ ID NO:200, which are parental sequences. The aptamer composition may comprise at least one RNA oligonucleotide truncated from the group consisting of SEQ ID NO:1 to SEQ ID NO:10, which are parental sequences. The aptamer composition may comprise at least one DNA oligonucleotide truncated from the group consisting of SEQ ID NO:101 to SEQ ID NO:110, which are parental sequences. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200, which is a parental sequence, wherein one or more nucleotides are deleted from the truncated oligonucleotide sequence. If one or more nucleotides are deleted from the truncated oligonucleotide sequence, the truncated oligonucleotide shows less than 100% sequence identity compared to the corresponding portion of the parental sequence. Suitable deletions do not alter the truncated secondary structure. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein the deletion from the truncated sequence does not exceed 10 nucleotides. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein the deletion from the truncated sequence does not exceed 5 nucleotides. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein the deletion from the truncated sequence does not exceed 4 nucleotides. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein no more than 3 nucleotides are deleted from the truncated sequence. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein no more than 2 nucleotides are deleted from the truncated sequence. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein 1 nucleotide is deleted from the truncated oligonucleotide sequence.

The aptamer composition may comprise at least one oligonucleotide forming the secondary structure disclosed herein and truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein one or more nucleotides are inserted into the truncated oligonucleotide sequence. If one or more nucleotides are inserted into the truncated oligonucleotide sequence, the truncated oligonucleotide exhibits less than 100% sequence identity compared to the corresponding portion of the parental sequence. Suitable insertions do not alter the secondary structure of the truncated oligonucleotide. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein no more than 10 nucleotides are inserted into the truncated sequence. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein no more than 5 nucleotides are inserted into the truncated sequence. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein no more than 4 nucleotides are inserted into the truncated sequence. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein no more than 3 nucleotides are inserted into the truncated sequence. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein no more than 2 nucleotides are inserted into the truncated sequence. The aptamer composition may comprise at least one oligonucleotide truncated from an oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200 as a parental sequence, wherein 1 nucleotide is inserted into the truncated oligonucleotide sequence.

The truncated oligonucleotide of the aptamer composition may comprise at least one oligonucleotide selected from the group consisting of at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO:201 to SEQ ID NO:212. The truncated oligonucleotide of the aptamer composition may comprise at least one oligonucleotide containing at least 10 adjacent nucleotides of a sequence selected from the group consisting of oligonucleotides of SEQ ID NO:201 to SEQ ID NO:212. At least one oligonucleotide may contain one or more motifs selected from the group consisting of: SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211 and SEQ ID NO:212. The aptamer composition may comprise at least one oligonucleotide comprising a nucleotide sequence having at least 80% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211 and SEQ ID NO:212. The aptamer composition may comprise at least one oligonucleotide comprising a nucleotide sequence having at least 90% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211 and SEQ ID NO:212. The aptamer composition may comprise at least one oligonucleotide comprising a nucleotide sequence having at least 95% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211 and SEQ ID NO:212.

The aptamer composition may comprise at least one oligonucleotide of RNA oligonucleotide, the at least one oligonucleotide forming the disclosed secondary structure and truncated from the group consisting of SEQ ID NO:1 to SEQ ID NO:10 as parental sequences. The aptamer composition may comprise at least one oligonucleotide truncated from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8 as parental sequences. The aptamer composition may comprise at least one oligonucleotide comprising a nucleotide sequence having at least 80% nucleotide sequence identity with the sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8 as a parental sequence. The aptamer composition may comprise at least one oligonucleotide having at least 90% nucleotide sequence identity with a parental sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8. The aptamer composition may comprise at least one oligonucleotide having at least 93% nucleotide sequence identity with a parental sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8. The aptamer composition may comprise at least one oligonucleotide having at least 96% nucleotide sequence identity with a parental sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8. The aptamer composition may comprise at least one oligonucleotide that has at least 99% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:8 as a parental sequence.

The aptamer composition may comprise at least one oligonucleotide of DNA oligonucleotide, the at least one oligonucleotide forming the disclosed secondary structure and truncated from the group consisting of SEQ ID NO:101 to SEQ ID NO:110 as parental sequences. The aptamer composition may comprise at least one oligonucleotide truncated from the group consisting of SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107 and SEQ ID NO:108 as parental sequences. The aptamer composition may comprise at least one oligonucleotide comprising a nucleotide sequence having at least 80% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107 and SEQ ID NO:108 as a parental sequence. The aptamer composition may comprise at least one oligonucleotide having at least 90% nucleotide sequence identity with a parental sequence selected from the group consisting of SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:108. The aptamer composition may also comprise at least one oligonucleotide having at least 93% nucleotide sequence identity with a parental sequence selected from the group consisting of SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:108. The aptamer composition may comprise at least one oligonucleotide having at least 96% nucleotide sequence identity with a parental sequence selected from the group consisting of SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:108. The aptamer composition may also comprise at least one oligonucleotide having at least 99% nucleotide sequence identity with a parental sequence selected from the group consisting of SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:108.

The aptamer compositions disclosed herein may comprise at least one oligonucleotide forming the disclosed secondary structure and selected from the group consisting of oligonucleotides selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246, or may comprise at least one oligonucleotide showing at least 80% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246. The aptamer compositions may comprise at least one oligonucleotide showing at least 90% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246. The aptamer compositions may comprise at least one oligonucleotide showing at least 95% nucleotide sequence identity with a sequence selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246. The aptamer composition may be selected from the group consisting of SEQ ID NO:237 to SEQ ID NO:246 and/or from oligonucleotide sequences showing at least 80% or at least 90% or at least 95% nucleotide sequence identity with the group consisting of SEQ ID NO:237 to SEQ ID NO:246.

In one respect, the aptamer composition has binding affinity for human intercellular adhesion molecule 1 (ICAM-1) (SEQ ID NO:213), its natural variants, polymorphic variants, or any post-translational modified form of the protein. Non-limiting examples of post-translational modifications of ICAM-1 include disulfide bonds (e.g., between Cys48 and Cys92, between Cys52 and Cys96, between Cys135 and Cys186, between Cys237 and Cys290, between Cys332 and Cys371, between Cys403 and Cys419, between Cys431 and Cys457), glycosylation (e.g., at Asn130, Asn145, Asn183, Asn202, Asn267, Asn296, Asn385, and Asn406), phosphorylation (e.g., at Thr521 or Thr530), and ubiquitination.

In one aspect, the aptamer composition has binding affinity for the extracellular domain of human intercellular adhesion molecule 1 (ICAM-1) (SEQ ID NO: 214) or any post-translational modification of said domain. In another aspect, the aptamer composition has binding affinity for one or more domains of intercellular adhesion molecule 1 (ICAM-1), said domain being selected from the group consisting of: Ig-like C2 type 1 domain (SEQ ID NO: 215), Ig-like C2 type 2 domain (SEQ ID NO: 216), Ig-like C2 type 3 domain (SEQ ID NO: 217), Ig-like C2 type 4 domain (SEQ ID NO: 218), Ig-like C2 type 5 domain (SEQ ID NO: 219), any post-translational modification of said domain, and mixtures thereof. On one hand, the aptamer composition has binding affinity for the Ig-like C2 type 1 domain (SEQ ID NO: 215) of intercellular adhesion molecule 1 (ICAM-1), any post-translational modifications of said domain, and mixtures thereof.

In one aspect, the aptamer composition has binding affinity for one or more regions of human intercellular adhesion molecules 1, 2, 3, or 4, wherein said regions comprise an amino acid sequence selected from the group consisting of SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and fragments thereof.

Chemical modification can introduce new features into aptamers, such as interactions with different molecules of the target, improved binding affinity, enhanced oligonucleotide conformational stability, or increased nuclease resistance. In one aspect, at least one oligonucleotide in the aptamer composition may contain native or non-native nucleobases. Native nucleobases are adenine, cytosine, guanine, thymine, and uracil. Non-limiting examples of non-natural nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thion-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosine-1-yl, 2-aminoadenine-9-yl, 7-deazo-7-iodoadenine-9-yl, 7-deazo-7-propynyl-2-aminoadenine-9-yl, phenoxazinyl, phenoxazinyl-G-clam, bromouracil, 5-iodouracil, and mixtures thereof.

Modification of the phosphate backbone of oligonucleotides can also increase resistance to nuclease digestion. On one hand, the nucleosides of oligonucleotides can be linked by chemical motifs selected from the group consisting of: native phosphate diesters, chiral thiophosphates, chiral methyl phosphonates, chiral aminophosphates, chiral phosphates, chiral triesters, chiral borophosphates, chiral selenophosphates, dithiophosphates, thioaminophosphates, methylene methylimino, 3'-amides, 3'-chiral aminophosphates, 3'-chiral methylene phosphonates, thiomethyl acetals, diethyl sulfide, fluorophosphates, and mixtures thereof. On the other hand, the nucleosides of oligonucleotides can be linked by native phosphate diesters.

On the one hand, the nucleotide sugar moiety of the oligonucleotide may be selected from the group consisting of: ribose, deoxyribose, 2'-fluorodeoxyribose, 2'-O-methylribose, 2'-O-(3-amino)propylribose, 2'-O-(2-methoxy)ethylribose, 2'-O-2-(N,N-dimethylaminooxy)ethylribose, 2'-O-2-[2-(N,N-dimethylamino)ethoxy]ethylribose, 2'-O-N,N-dimethylacetamidoribose, N-morpholinophosphonidyldiamine, α-deoxyfuranose, other pentoses, hexoses, and mixtures thereof.

On one hand, the derivatives of ribonucleotides or the derivatives of said deoxyribonucleotides may be selected from the group consisting of: oligonucleotides, peptide oligonucleotides, diol oligonucleotides, threonucleotides, hexitol oligonucleotides, azotol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabinose oligonucleotides, 2'-fluoroarabinose oligonucleotides, cyclohexene oligonucleotides, diaminophosphate morpholino oligonucleotides, and mixtures thereof.

In one aspect, the nucleotides at the 5' and 3' ends of at least one oligonucleotide may be reversed. In another aspect, at least one nucleotide of at least one oligonucleotide may be fluorinated at the 2' position of the pentose group. In another aspect, the pyrimidine nucleotide of said at least one oligonucleotide may be fluorinated at the 2' position of the pentose group. In one aspect, the aptamer composition may comprise at least one polymer material, wherein said at least one polymer material is covalently linked to said at least one oligonucleotide. In another aspect, said at least one polymer material may be polyethylene glycol.

In one aspect, the length of the at least one oligonucleotide may be between about 30 nucleotides and about 60 nucleotides. In another aspect, the length of the at least one oligonucleotide may be less than about 50 nucleotides, and alternatively, the length of the at least one oligonucleotide may be less than about 40 nucleotides.

In one aspect, the at least one oligonucleotide may be covalently or non-covalently attached to one or more additional active ingredients. In another aspect, the one or more active ingredients may be selected from the group consisting of: respiratory disease treatments, cold treatments, influenza treatments, antiviral agents, antimicrobial agents, cooling agents, warming agents, odor absorbers, natural extracts, peptides, enzymes, pharmaceutical active ingredients, metal compounds, and mixtures thereof. In another aspect, the one or more active ingredients may include, but are not limited to, pharmaceutical active ingredients, menthol, L-menthol, zinc and its salts, eucalyptus, camphor, and combinations thereof. Suitable active ingredients include any material generally considered safe and providing beneficial healthcare effects.

In one aspect, the at least one oligonucleotide can be non-covalently attached to the one or more active ingredients via molecular interactions. Examples of molecular interactions are electrostatic forces of aromatic rings, van der Waals interactions, hydrogen bonding, and π-π stacking.

In one aspect, one or more adapters or spacers can be used to covalently attach the at least one oligonucleotide to the one or more active ingredients. Non-limiting examples of adapters are chemically unstable adapters, enzymatically unstable adapters, and non-cleavable adapters. Examples of chemically unstable adapters are acid-cleavable adapters and disulfide adapters. Acid-cleavable adapters utilize low pH to trigger the hydrolysis of acid-cleavable bonds such as hydrazone bonds to release the active ingredient or payload. Disulfide adapters can release the active ingredient under reducing conditions. Examples of enzymatically unstable adapters are peptide adapters that can be cleaved in the presence of proteases and β-glucuronide adapters that are cleaved by glucuronidases that release the payload. Non-cleavable adapters can also release the active ingredient if the aptamer is degraded by a nuclease.

In one aspect, the at least one oligonucleotide may be covalently or non-covalently attached to one or more nanomaterials. In this invention, the at least one oligonucleotide and the one or more active ingredients may be covalently or non-covalently attached to one or more nanomaterials. In another aspect, the one or more active ingredients may be carried by the one or more nanomaterials. Non-limiting examples of nanomaterials may include gold nanoparticles, nanoscale iron oxides, carbon nanomaterials (such as single-walled carbon nanotubes and graphene oxides), mesoporous silica nanoparticles, quantum dots, liposomes, poly(lactide-co-glycolic acid) nanoparticles, polymer micelles, dendritic polymers, serum albumin nanoparticles, DNA-based nanomaterials, and combinations thereof. These nanomaterials can be used as carriers of bulky active ingredients, while aptamers can facilitate the delivery of nanomaterials containing active substances to the intended target.

Nanomaterials can have a variety of shapes or morphologies. Non-limiting examples of shapes or morphologies may include spheres, rectangles, polygons, disks, rings, cones, pyramids, rods/cylinders, and fibers. In the context of this invention, nanomaterials generally have at least one spatial dimension of less than about 100 μm, and more preferably less than about 10 μm. Nanomaterials include solid, semi-solid, or liquid materials.

Aptamers can also be peptides that bind to targets with high affinity and specificity. These peptide aptamers can be part of a scaffold protein. Peptide aptamers can be isolated from a combinatorial library and improved through directed mutagenesis or multiple rounds of variable region mutagenesis and selection. In one aspect, the aptamer composition may comprise at least one peptide or protein; wherein the aptamer composition has binding affinity for one or more cell membrane glycoproteins, wherein the one or more cell membrane glycoproteins may be selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), members of the low-density lipoprotein receptor (LDLR) family, and members of the cadherin-associated family 3 (CDHR3); preferably intercellular adhesion molecule 1 (ICAM-1), and wherein the aptamer is configured to reduce the binding of one or more human rhinoviruses to the cell membrane glycoprotein, preferably intercellular adhesion molecule 1 (ICAM-1). In particular, the aptamer composition may comprise at least one peptide or protein translated from at least one oligonucleotide disclosed herein.

III. Methods for Designing Parental Aptamer Compositions

The method for designing nucleic acid aptamers is known as Systematic Evolution of Ligands with Exponential Enrichment (SELEX), which has been extensively studied and improved for selecting aptamers for small molecules and proteins (WO 91/19813). In short, in the conventional form of SELEX, the process begins with the synthesis of a large library of oligonucleotides consisting of randomly generated, fixed-length sequences flanked by constant 5' and 3' ends that act as primers. The oligonucleotides in the library are then exposed to target ligands, and those that do not bind to the target are removed. The bound sequences are eluted and amplified by PCR (polymerase chain reaction) to prepare for subsequent rounds of selection, where the stringency of the elution conditions is typically increased to identify the most tightly bound oligonucleotides. In addition to conventional SELEX, improved forms exist, such as capillary electrophoresis SELEX, magnetic bead-based SELEX, cellular SELEX, automated SELEX, and composite target SELEX. A review of aptamer screening methods can be found in (1) Kim, Y.S. and M.B. Gu, “Advances in Aptamer Screening and Small Molecule Aptasensors”, Adv. Biochem. Eng. Biotechnol. 2014 140:29-67 (Biosensors based on Aptamers and Enzymes) and (2) Stoltenburg, R. et al. (2007) “SELEX - A(r)evolutionary method to generate high-affinity nucleic acid ligands”, Biomol. Eng. 2007 24(4):381-403, the contents of which are incorporated herein by reference. Although the SELEX method has been widely used, it is neither predictive nor standardized for each target. Instead, a method must be developed for each specific target to produce viable aptamers.

Despite the extensive selection of aptamers, SELEX has not been routinely used to select aptamers that possess binding affinity for cell membrane glycoproteins such as intercellular adhesion molecule-1 (ICAM-1), members of the low-density lipoprotein receptor (LDLR) family, and cadherin-associated family member 3 (CDHR3), and that prevent human rhinovirus from binding to these proteins. Unexpectedly, the inventors have discovered that SELEX can be used in the design of parental aptamers that prevent human rhinovirus from binding to the ICAM-1 receptor.

Select Library

In SELEX, the initial candidate library is typically a mixture of chemically synthesized DNA oligonucleotides, each containing a long variable region of n nucleotides. This long variable region has conserved regions or primer recognition regions on both sides of the 3' and 5' ends for all candidates in the library. These primer recognition regions allow for manipulation of the central variable region specifically via PCR during SELEX.

The length of the variable region determines the diversity of the library, which is equal to ⁴ⁿ , since each position can be occupied by one of four nucleotides: A, T, G, or C. Long variable regions result in enormous library complexity. For example, when n = 50, the theoretical diversity is ^4.50 or ^10.30 , which are practically inaccessible values, as this corresponds to over 10⁵ tons of material in the library, where each sequence is represented only once. The experimental limit is approximately ^10¹⁵ different sequences, which is the library containing all candidate sequences representing a variable region of 25 nucleotides. If a library with a variable region of 30 nucleotides containing a theoretical diversity of approximately ^10¹⁸ is chosen, only a 1/1000 probability will be explored. In practice, this is usually sufficient to obtain aptamers with the desired properties. Furthermore, because the polymerases used are unreliable and introduce errors at a rate of approximately ^10⁻⁴ , they contribute to significantly enriching the diversity of the sequence pool throughout the SELEX process. For a library of random regions of 100 nucleotides in length, one candidate out of the 100 will be modified in each amplification cycle, resulting in 10^ ¹³ new candidates for the entire library.

In one aspect, the starting mixture of oligonucleotides may contain more than about ^10⁶ different oligonucleotides, and more preferably between about ^10¹³ and about ^10¹⁵ different oligonucleotides. In another aspect, the length of the variable region may be between about 10 and about 100 nucleotides. In another aspect, the length of the variable region may be between about 20 and about 60 nucleotides. In another aspect, the length of the variable region may be about 40 nucleotides. Random regions shorter than 10 nucleotides may be used, but their ability to form secondary or tertiary structures and their ability to bind target molecules may be limited. Random regions longer than 100 nucleotides may also be used, but there may be difficulties in terms of synthesis cost. The randomness of the variable region is not a limitation of the invention. For example, if prior knowledge exists regarding oligonucleotides that bind to a given target, a library having such a sequence may be used as, or is superior to, a completely random library.

When designing primer recognition sequences, care should be taken to minimize potential annealing between sequences, annealing of post-folded regions within sequences, or annealing of the same sequence itself. On one hand, the length of primer recognition sequences can be between about 10 and about 40 nucleotides. On another hand, the length of primer recognition sequences can be between about 12 and about 30 nucleotides. On yet another hand, the length of primer recognition sequences can be between about 18 and about 26 nucleotides, i.e., about 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides. The length and sequence of the primer recognition sequences determine their annealing temperatures. On one hand, the primer recognition sequences of the oligonucleotides can have annealing temperatures between about 60°C and about 72°C.

Aptamers can be ribonucleotides (RNA), deoxynucleotides (DNA), or derivatives thereof. When the aptamer is a ribonucleotide, the first SELEX step may include an initial mixture of chemically synthesized DNA oligonucleotides transcribed at the 5' end via primer recognition sequences. After selection, the candidates are converted back to DNA by reverse transcription before amplification. RNA and DNA aptamers with comparable properties have been selected for the same targets and are reported in the art. Furthermore, these two types of aptamers can be competitive inhibitors of each other, indicating potential overlap in interaction sites.

New functions, such as hydrophobicity or photoreactivity, can be incorporated into oligonucleotides by modifying the nucleobases before or after selection. Modifications at the C-5 position of pyrimidines or at the C-8 or N-7 position of purines are particularly common and are compatible with certain enzymes used during the SELEX amplification step. In one aspect, the oligonucleotides may contain native or non-native nucleobases. Native nucleobases include adenine, cytosine, guanine, thymine, and uracil. Non-limiting examples of non-natural nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thion-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosine-1-yl, 2-aminoadenine-9-yl, 7-deazo-7-iodoadenine-9-yl, 7-deazo-7-propynyl-2-aminoadenine-9-yl, phenoxazinyl, phenoxazinyl-G-clam, 5-bromouracil, 5-iodouracil, and mixtures thereof. Some non-natural nucleobases, such as 5-bromouracil or 5-iodouracil, can be used to generate photocrosslinkable aptamers that can be activated by UV light to form a covalent bond with a target.

In one aspect, the nucleosides of the oligonucleotide can be linked by chemical motifs selected from the group consisting of: native phosphodiester, chiral thiophosphate, chiral methyl phosphonate, chiral aminophosphate, chiral phosphate chiral triester, chiral borophosphate, chiral selenophosphate, dithiophosphate, thioaminophosphate, methylene methylimino, 3'-amide, 3'-chiral aminophosphate, 3'-chiral methylene phosphonate, thiomethyl acetal, diethyl sulfide, fluorophosphate, and mixtures thereof. In another aspect, the nucleosides of the oligonucleotide can be linked by native phosphodiester.

In one respect, the glycosylated portion of the oligonucleotide may be selected from the group consisting of: ribose, deoxyribose, 2'-fluorodeoxyribose, 2'-O-methylribose, 2'-O-(3-amino)propylribose, 2'-O-(2-methoxy)ethylribose, 2'-O-2-(N,N-dimethylaminooxy)ethylribose, 2'-O-2-[2-(N,N-dimethylamino)ethoxy]ethylribose, 2'-O-N,N-dimethylacetamidoribose, N-morpholinophosphonidyldiamine, α-deoxyfuranose, other pentoses, hexoses, and mixtures thereof.

In one aspect, the derivative of the ribonucleotide or the derivative of the deoxyribonucleotide may be selected from the group comprising: oligonucleotides, peptide oligonucleotides, diol oligonucleotides, threonucleotides, hexitol oligonucleotides, azotol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabinose oligonucleotides, 2'-fluoroarabinose oligonucleotides, cyclohexene oligonucleotides, diaminophosphate morpholino oligonucleotides, and mixtures thereof.

When modified nucleotides are used in the SELEX process, they should be compatible with the enzymes used during the amplification step. Non-restrictive examples of modifications compatible with commercial enzymes include modifications at the 2' position of the sugar in the RNA library. The 2'-OH group of the ribose in pyrimidine nucleotides can be replaced with 2'-amino, 2'-fluorine, 2'-methyl, or 2'-O-methyl groups, which protect the RNA from nuclease degradation. Additional modifications to the phosphate linker, such as thiophosphate and borophosphate, are also compatible with polymerases and confer nuclease resistance.

In one aspect, at least one nucleotide of the oligonucleotide may be fluorinated at the 2' position of the pentose group. In another aspect, the pyrimidine nucleotide of the oligonucleotide may be at least partially fluorinated at the 2' position of the pentose group. In another aspect, all pyrimidine nucleotides of the oligonucleotide may be fluorinated at the 2' position of the pentose group. In another aspect, at least one nucleotide of the oligonucleotide may be amination at the 2' position of the pentose group.

Another approach, recently described as two-dimensional SELEX, simultaneously applies in vitro oligonucleotide selection and dynamic combinatorial chemistry (DCC), such as reversible reactions between certain groups (amines) of oligonucleotides and a library of aldehyde compounds. This reaction produces imine oligonucleotides selected on the same principle as conventional SELEX. Therefore, aptamers targeting hairpin RNA modifications that differ from natural aptamers can be identified.

A very different approach involves the use of optical isomers. Natural oligonucleotides are D-isomers. L-analytes are nuclease resistant but cannot be synthesized by polymerases. According to the rules of optical isomerism, L-series aptamers can form complexes with their targets (T) that have the same properties as complexes formed by D-series isomers and their enantiomers (T') of the target (T). Therefore, if compound T' is chemically synthesizable, it can be used for the selection of natural aptamers (D). Once identified, this aptamer can be chemically synthesized as an L-series aptamer. This L-aptamer is a ligand for the natural target (T).

Select Steps

Single-stranded oligonucleotides can fold to produce secondary and tertiary structures, similar to base pair formation. Therefore, the initial sequence library is a library of three-dimensional shapes, each corresponding to a distribution of units that can trigger electrostatic interactions, generate hydrogen bonds, etc. Selection becomes a problem of identifying shapes within the library suitable for the target—that is, shapes that allow for the maximum number of interactions and the formation of the most stable aptamer-target complex. For small targets (dyes, antibiotics, etc.), the identified aptamers are characterized by equilibrium dissociation constants in the micromolar range, while for protein targets, Kd values below ^10⁻⁹ M are not uncommon.

Selection in each round is performed by physically separating the target-associated oligonucleotide from the free oligonucleotides. Various techniques can be applied (chromatography, filtration retention, electrophoresis, etc.). Selection conditions (relative concentration of target/candidate, ion concentration, temperature, washing, etc.) are adjusted to induce target-binding competition among the oligonucleotides. Generally, the stringency increases with each round to facilitate the capture of the oligonucleotide with the highest affinity. Furthermore, anti-selection or negative selection is performed to eliminate oligonucleotides that recognize the carrier or unwanted targets (e.g., filters, beads, etc.).

The SELEX process for selecting target-specific parental aptamers is characterized by repeating five main steps: (1) binding oligonucleotides to the target, (2) dispensing or removing oligonucleotides with low binding affinity, (3) eluting oligonucleotides with high binding affinity, (4) amplifying or replicating oligonucleotides with high binding affinity, and (5) regulating or preparing oligonucleotides for the next cycle. This selection process is designed to identify oligonucleotides with maximum affinity and specificity for the target material.

In one aspect, a method for designing a composition from which a parental aptamer can be truncated to form the desired secondary structure of the aptamer disclosed herein may include the steps of contacting: a) a mixture of oligonucleotides, b) a selection buffer, and c) a target material comprising one or more cell membrane glycoproteins selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), members of the low-density lipoprotein receptor (LDLR) family, members of the cadherin-associated family 3 (CDHR3), truncated forms thereof, and mixtures thereof; preferably intercellular adhesion molecule 1 (ICAM-1) and truncated forms thereof. In another aspect, a method for designing a composition of the parental aptamer disclosed herein may include the steps of contacting: a) a mixture of oligonucleotides, b) a selection buffer, and c) a cell expressing one or more cell membrane glycoproteins selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), members of the low-density lipoprotein receptor (LDLR) family, members of the cadherin-associated family 3 (CDHR3), truncated forms thereof, and mixtures thereof; preferably intercellular adhesion molecule 1 (ICAM-1) and truncated forms thereof. On another aspect, a method for designing compositions of the parental aptamers disclosed herein may include the steps of contacting the following substances: a) a mixture of oligonucleotides, b) a selection buffer, and c) human nasal epithelial cells expressing one or more cell membrane glycoproteins selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), members of the low-density lipoprotein receptor (LDLR) family, members of the cadherin-associated family 3 (CDHR3), truncated forms thereof, and mixtures thereof; preferably intercellular adhesion molecule 1 (ICAM-1) and truncated forms thereof.

In one aspect, the mixture of oligonucleotides may include oligonucleotides selected from the group consisting of deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof. Furthermore, the one or more cell membrane glycoproteins or truncated forms thereof may be mixed and separated with other substances such as proteins or peptides or a portion of the cell expressing the glycoprotein.

The SELEX cycle is typically repeated several times until an oligonucleotide with high binding affinity is identified. The number of cycles depends on several variables, including target characteristics and concentration, the design of the initial random oligonucleotide library, selection conditions, the ratio of target binding sites to oligonucleotides, and the efficiency of the partitioning step. In one aspect, the contacting step may be performed at least five times. In another aspect, the contacting step may be performed between six and 30 times. In yet another aspect, the method may further include a step of removing oligonucleotides that do not bind to the target material during the contacting step.

Oligonucleotides are oligoanions, each unit possessing a charge and hydrogen bond donor/acceptor site at a specific pH. Therefore, the pH and ionic strength of the selection buffer are important and should represent the conditions for the intended aptamer application. In one aspect, the pH of the selection buffer may be between about 2 and about 9, alternatively between about 5 and about 8.

Cations not only facilitate the proper folding of oligonucleotides but also provide beneficial effects. In one aspect, the selection buffer may contain cations. Non-limiting examples of cations are Na ⁺ , K ⁺ , Mg2 ⁺ , and Ca2 ⁺ .

To ensure that the aptamer retains its structure and function during its application, the in vitro selection process can be performed under conditions similar to those being developed. In one aspect, the selection buffer may comprise a solution or suspension of a personal healthcare composition selected from the group consisting of: tablets, lyophilized tablets, lollipops, tablets, liquid-centered candies, confectionery, powders, particulate matter, membranes, liquids, solutions, suspensions, oral rinsings or mouthwashes, saline washes, dispersible fluids, sprays, rapidly dissolving fibers, vapors, creams, ointments, powders, particulate matter, membranes, and combinations thereof.

In one aspect, the selection buffer may comprise at least one surfactant. In another aspect, the at least one surfactant may be selected from the group consisting of anionic surfactants, amphoteric or zwitterionic surfactants, and mixtures thereof. Non-limiting examples of anionic surfactants are alkyl and alkyl ether sulfates or sulfonates, including ammonium lauryl sulfate, ammonium lauryl polyoxyethylene ether sulfate, triethylamine lauryl sulfate, triethylamine lauryl polyoxyethylene ether sulfate, triethanolamine lauryl sulfate, triethanolamine lauryl polyoxyethylene ether sulfate, monoethanolamine lauryl sulfate, monoethanolamine lauryl polyoxyethylene ether sulfate, diethanolamine lauryl sulfate, diethanolamine lauryl polyoxyethylene ether sulfate, sodium monoglyceride lauryl sulfate, sodium lauryl sulfate, and lauryl poly(alkyl) ether sulfate. Sodium oxyethylene ether sulfate, potassium lauryl sulfate, potassium lauryl polyoxyethylene ether sulfate, sodium lauryl sarcosinate, sodium lauroyl sarcosinate, lauryl sarcosinate, cocoyl sarcosinate, ammonium cocoyl sulfate, ammonium lauroyl sulfate, sodium cocoyl sulfate, sodium lauroyl sulfate, potassium cocoyl sulfate, potassium lauryl sulfate, triethanolamine lauryl sulfate, triethanolamine lauryl sulfate, monoethanolamine cocoyl sulfate, monoethanolamine lauryl sulfate, sodium tridecylbenzenesulfonate, sodium dodecylbenzenesulfonate, sodium cocoyl hydroxyethanesulfonate, and combinations thereof. Non-limiting amphoteric surfactants include those surfactants broadly described as derivatives of aliphatic secondary and tertiary amines, wherein the aliphatic group may be linear or branched, and wherein one of the aliphatic substituents comprises about 8 to about 18 carbon atoms, and one of the aliphatic substituents comprises an anionic group, such as a carboxyl, sulfonate, sulfate, phosphate, or phosphonate, including cocoamphoacetate, cocoamphodiacetate, lauroylamphoacetate, lauroylamphodiacetate, and mixtures thereof. Non-limiting examples of amphoteric surfactants include those surfactants broadly described as derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds, wherein the aliphatic group may be linear or branched, and wherein one of the aliphatic substituents comprises about 8 to about 18 carbon atoms, and one of the aliphatic substituents comprises an anionic group, such as a carboxyl, sulfonate, sulfate, phosphate, or phosphonate and betaine.

The buffer solution may contain at least one material selected from the group consisting of: aqueous carriers, gel matrices, siloxane conditioners, organic conditioners, nonionic polymers, deposition aids, rheology modifiers/suspending agents, beneficial agents, and mixtures thereof. Non-limiting examples of aqueous carriers include water and aqueous solutions of lower alkyl alcohols and polyols (including ethanol, isopropanol, propylene glycol, hexane glycol, glycerol, and propylene glycol). Non-limiting examples of gel matrices include aqueous solutions of fatty alcohols, including cetyl alcohol, stearyl alcohol, docosyl alcohol, and mixtures thereof. Non-limiting examples of siloxane conditioners include polydimethylsiloxane, polydimethylsiloxane alcohol, cyclic siloxanes, methylphenyl polysiloxane, and modified siloxanes having various functional groups such as amino groups, quaternary ammonium salt groups, aliphatic groups, alcohol groups, carboxylic acid groups, ether groups, sugar or polysaccharide groups, fluorinated alkyl groups, alkoxy groups, or combinations of such groups. Non-limiting examples of organic conditioning materials include hydrocarbon oils, polyolefins, fatty esters, fluorinated conditioning compounds, fatty alcohols, alkyl glucosides and alkyl glucoside derivatives, quaternary ammonium compounds, polyethylene glycols and polypropylene glycols (including those with CTFA names of PEG-200, PEG-400, PEG-600, PEG-1000, PEG-2M, PEG-7M, PEG-14M, and PEG-45M) having a molecular weight of up to about 2,000,000, and mixtures thereof. Non-limiting examples of nonionic polymers include polyalkylene glycols, such as polyethylene glycols. Non-limiting examples of deposition aids include copolymers of vinyl monomers having cationic amine or quaternary ammonium functional groups with water-soluble spacer monomers (such as acrylamide, methacrylamide, alkyl and dialkyl acrylamides, alkyl and dialkyl methacrylamides, alkyl acrylates, alkyl methacrylates, vinyl caprolactone, and vinylpyrrolidone); vinyl esters, vinyl alcohol (prepared by hydrolysis of polyvinyl acetate), maleic anhydride, propylene glycol and ethylene glycol, cationic cellulose, cationic starch, and cationic guar gum. Non-limiting examples of rheology modifiers/suspending agents include homopolymers based on acrylic acid, methacrylic acid, or other related derivatives, alginate-based materials, and cellulose derivatives. Non-limiting examples of beneficial agents include whitening agents, fortifying agents, antifungal agents, antibacterial agents, antimicrobial agents, antidandruff agents, antiodor agents, fragrances, olfactory enhancers, antipruritic agents, cooling agents, anti-adhesion agents, moisturizers, smoothing agents, surface modifiers, antioxidants, natural extracts and essential oils, dyes, pigments, bleaching agents, nutrients, peptides, vitamins, enzymes, chelating agents, and mixtures thereof.

Negative selection or anti-selection steps can minimize the enrichment of oligonucleotides that bind to undesirable targets or undesirable epitopes within targets. In one aspect, the method for designing parental aptamer compositions may further include the step of contacting: a) a mixture of oligonucleotides, b) a selection buffer, and c) one or more undesirable targets. Methods for negative selection or anti-selection of aptamers against unbound targets are disclosed in WO201735666, the contents of which are incorporated herein by reference.

A method for designing parental aptamer compositions may include the following steps: a) synthesizing a mixture of oligonucleotides; b) contacting i. the mixture of oligonucleotides, ii. a selection buffer, and iii. a target material comprising one or more cell membrane glycoproteins; wherein the glycoproteins are selected from the group consisting of intercellular adhesion molecule 1 (ICAM-1), fragments thereof, and combinations thereof, to generate a target suspension; c) removing the liquid phase from the target suspension to generate a target-oligonucleotide mixture; d) contacting the target-oligonucleotide mixture with a washing buffer and removing the liquid phase to generate a target-aptamer mixture; and e) contacting the target-aptamer mixture with an elution buffer and recovering the liquid phase to generate an aptamer mixture. In one aspect, the steps may be repeated at least five times. In another aspect, the steps may be performed between six and 30 times, preferably less than 20 times.

On the other hand, a method for designing a parent aptamer composition includes the following steps: a) synthesizing a random mixture of deoxyribonucleotides comprising oligonucleotides, the oligonucleotides being composed of: i. a T7 promoter sequence at the 5' end, ii. a variable 40-nucleotide sequence in the middle, and iii. a conserved reverse primer recognition sequence at the 3' end; b) transcribing the random mixture of the deoxyribonucleotides using a pyrimidine nucleotide fluorinated at the 2' position of the pentose group and a native purine nucleotide, and a mutant T7 polymerase to produce a mixture of fluorinated ribonucleotides; c) contacting: i. the mixture of fluorinated ribonucleotides, ii. a selection buffer, and iii. a mixture containing one or more cells. The target material for the membrane glycoprotein; wherein the glycoprotein is selected from the group consisting of: intercellular adhesion molecule 1 (ICAM-1), fragments thereof, and combinations thereof, to generate a target suspension; d) removing the liquid phase from the target suspension to generate a target-oligonucleotide mixture; e) contacting the target-oligonucleotide mixture with a washing buffer and removing the liquid phase to generate a target-aptamer mixture; f) contacting the target-aptamer mixture with an elution buffer and recovering the liquid phase to generate an RNA aptamer mixture; g) reverse transcription and amplification of the RNA aptamer mixture to generate a DNA copy of the RNA aptamer mixture; and h) sequencing the DNA copy of the RNA aptamer mixture.

Modifications after selection

To improve the stability of the aptamer, chemical modifications can be introduced into the aptamer after the selection process. For example, the 2'-OH group of the ribose moiety can be replaced with 2'-fluorine, 2'-amino, or 2'-O-methyl. Furthermore, the 3' and 5' ends of the aptamer can be capped with different groups, such as streptavidin-biotin, reverse thymidine, amines, phosphate groups, polyethylene glycol, cholesterol, fatty acids, proteins, enzymes, fluorophores, etc., thereby endowing the oligonucleotide with exonuclease resistance or providing additional beneficial effects. Other modifications are described in previous sections of this disclosure.

Unlike backbone modification, which can lead to loss of aptamer-target interaction properties, it is possible to conjugate various groups at either the 3' or 5' end of an oligonucleotide to transform it into a delivery vector, tool, probe, or sensor without compromising its properties. This flexibility constitutes a significant advantage of aptamers, especially in their application in this invention. In one aspect, one or more personal care active ingredients may be covalently attached to the 3' end of said at least one oligonucleotide. In another aspect, one or more personal care active ingredients may be covalently attached to the 5' end of said at least one oligonucleotide. In yet another aspect, one or more personal care active ingredients may be covalently attached to random positions of said at least one oligonucleotide.

The incorporation of modifications to aptamers can be performed using enzymatic or chemical methods. Non-limiting examples of enzymes used for modifying aptamers include terminal deoxynucleotidyl transferase (TdT), T4 RNA ligase, T4 polynucleotide kinase (PNK), DNA polymerase, RNA polymerase, and other enzymes known to those skilled in the art. TdT is a template-independent polymerase that can add modified deoxynucleotides to the 3' end of deoxyribonucleotides. T4 RNA ligase can be used to label ribonucleotides at the 3' end using appropriately modified 3',5'-bisphosphonates. PNK can be used to phosphorylate the 5' end of synthetic oligonucleotides, thereby enabling them to undergo further chemical transformations (see below). DNA polymerases and RNA polymerases are commonly used to randomly incorporate modified nucleotides throughout the sequence, provided that the nucleotide is compatible with the enzyme.

Non-limiting examples of chemical methods for modifying aptamers include periodate oxidation of ribonucleotides, EDC activation of 5'-phosphate, random chemical labeling methods, and other chemical methods known to those skilled in the art and incorporated herein.

During periodate oxidation, metaperiodate and properiodate cleave the C-C bonds between adjacent diols of the 3'-ribonucleotide, producing two aldehyde moieties that allow for the conjugation of tags or active ingredients at the 3' end of RNA aptamers. The resulting aldehydes readily react with hydrazine- or primary amine-containing molecules. When using amines, the resulting Schiff base can be reduced to a more stable secondary amine using sodium cyanoborohydride (NaCNBH3).

When activated with EDC of 5'-phosphate, the 5'-phosphate of oligonucleotides is typically activated with EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and imidazole to generate a reactive imidazole intermediate, which is then reacted with a primary amine to produce an aptamer modified at the 5' end. Because the reaction requires a 5' phosphate ester group, the oligonucleotide can be synthesized first by treatment with a kinase (e.g., PNK).

Random chemical labeling can be performed using various methods. Because these methods allow labeling at random sites along the aptamer, a higher degree of modification can be achieved compared to end-labeling methods. However, the binding of the aptamer to its target can be disrupted due to the modification of the nucleobases. The most common random chemical modification methods involve the use of photoreactive reagents, such as those based on azidophenyl. When the azidophenyl group is exposed to UV light, it forms an unstable nitrobenzene, which reacts with the double bond of the aptamer as well as the C-H and N-H sites.

Additional information on methods for modifying aptamers is summarized in Hermanson G.T.’s “Bioconjugate Techniques” (pp. 969–1002, 2nd edition, Academic Press, San Diego, 2008), which is incorporated herein by reference.

Following selection, in addition to chemical modification, sequence truncation can be performed to remove regions not essential for binding or folding into a structure. Furthermore, aptamers can be linked together to provide different characteristics or better affinity. Therefore, any truncated or combined aptamers described herein can also be incorporated into aptamer compositions.

IV. Methods for identifying aptamers to be truncated from the parent aptamer

Suitable truncated sequences that form the appropriate secondary structures disclosed herein can be identified through theoretical analysis. Therefore, the main effective regions of the parental aptamer must be determined based on stable secondary structure formation. Typically, the parental aptamer varies between possible shapes at 37°C, and the possible shapes that a single sequence can form can be characterized, for example, through theoretical analysis using online software provided by the ViennaRNA Web Services created by the Theoretical Biology Group of the Institute for Theoretical Chemistry at the University of Vienna. In addition to the theoretical possibilities, the relative proportions of each shape at a given temperature can be calculated. Therefore, truncated aptamers that exhibit certain of these shapes with a high level of stability can be selected for further analysis.

V. Application of aptamer compositions in personal healthcare products

This document describes personal healthcare compositions and methods of using such compositions to prevent and treat cold-like symptoms caused by respiratory viral infections. In some aspects, the personal healthcare compositions comprise at least one aptamer as disclosed herein; wherein the at least one aptamer has binding affinity for ICAM-1 and is configured to reduce the binding of one or more human rhinoviruses to intercellular adhesion molecule 1 (ICAM-1). The personal healthcare compositions may preferably be applied to upper respiratory tract areas, such as the nasal cavity and pharynx, to provide a barrier against rhinovirus binding and entry into cells.

Personal healthcare compositions preferably contain at least one aptamer in a pharmaceutically effective amount disclosed herein. In some aspects, personal healthcare compositions may contain at least one aptamer in amounts of about 0.001% to about 1%, or about 0.005% to about 0.5%, or about 0.01% to about 0.1%, all percentages being by weight of the composition.

Personal healthcare compositions can be administered orally or intranasally. In one aspect, a personal healthcare composition can be an oral composition. Oral compositions can be in liquid, semi-solid, suspension, or any solid form capable of rapidly dissolving in the mouth. Non-limiting examples of oral dosage forms may include tablets, lyophilized tablets, lollipops, tablets, liquid-centered candies, candies, powders, particulate matter, films, liquids, solutions, suspensions, oral rinsings or mouthwashes, saline washes, dispersible fluids, sprays, rapidly dissolving fibers such as polyvinylpyrrolidone and poly(vinyl alcohol), and combinations thereof. Solid oral dosage forms can have any desired size, shape, weight, consistency, or hardness, taking into account that they should not be swallowed before disintegration and can be easily placed in the mouth. Alternatively, a personal healthcare composition can be a nasal composition. Nasal compositions can be any dosage form capable of rapidly dispersing in the nose. Non-limiting examples of nasal dosage forms may include vapors, creams, ointments, powders, particulate matter, films, liquids, dispersible fluids, sprays, and combinations thereof.

As used herein, the term “application” in relation to humans/mammals means the ingestion or directed ingestion, or consumption, or delivery, or chewing, or drinking or spraying, or placing in the mouth or nose, or inhalation of one or more personal healthcare compositions by a human/mammal. Application may be performed as needed or desired, such as once a week or daily, including multiple times a day, such as at least once a day, at least twice a day, at least three times a day, or at least four times a day.

Personal healthcare compositions can be used to prevent and treat cold-like symptoms. As used herein, “cold-like symptoms” refers to symptoms commonly associated with respiratory viral infections. These symptoms include, but are not limited to, nasal congestion, chest tightness, sneezing, runny nose, fatigue or malaise, cough, fever, sore throat, headache, and other known cold symptoms.

As further used herein, “treatment” in relation to respiratory diseases means any similar benefit of the application of the mentioned compositions to prevent, alleviate, improve, suppress, or reduce one or more symptoms of a respiratory disease or the respiratory disease itself, or to a mammalian subject in need, preferably a human, of a respiratory disease. Thus, such benefits include, for example: prevention of the occurrence of a respiratory disease or its associated symptoms in mammals, such as when a mammal is susceptible to a respiratory disease but has not yet been diagnosed with it; suppression of a respiratory disease or its associated symptoms; and/or alleviation, reversal, or cure of a respiratory disease or its associated symptoms. With regard to the method of the invention relating to the prevention of respiratory diseases, it should be understood that the term “prevention” does not require complete blocking of the respiratory disease. Rather, as used herein, the term “prevention,” etc., refers to the ability of a person skilled in the art to identify susceptibility to a respiratory disease (such as, for example, in humans during winter months) such that the application of the mentioned compositions can occur before the onset of disease-related symptoms.

The personal healthcare compositions and methods of the present invention may include, consist of, or consist essentially of the following elements: the essential elements and limitations of the invention as described herein, and any additional or optional ingredients, components, or limitations described herein or otherwise used in personal healthcare compositions intended for use by a subject.

Unless otherwise specified, all parts, percentages and ratios herein are by weight. Unless otherwise specified, all such weights relating to the listed ingredients are based on active substance levels and therefore do not include solvents or byproducts that may be included in commercially available materials. Unless otherwise specified, all measurements herein were performed at 25°C.

The personal healthcare composition of the present invention may comprise one or more of the following substances:

Personal healthcare compositions may contain solvents. Non-limiting examples of solvents include water, propylene glycol, ethanol, glycerin, polyethylene glycol, and mixtures thereof. The solvent may be present in an amount of about 2% to about 99%, about 5% to about 95%, about 10% to about 80%, about 12% to about 65%, or about 20% to about 50% by weight of the composition.

Personal healthcare compositions may contain thickeners. Non-limiting examples of thickeners may include carboxymethyl cellulose (CMC), sodium carboxymethyl cellulose, and mixtures thereof. When present, the composition may contain about 0.01% to about 60%, or about 0.1% to about 40%, or about 1% to about 30%, or about 2% to about 20%, or about 3% to about 15% of the thickener, all percentages being by weight of the composition. In one aspect, the thickener may provide moisturizing and/or hydrating benefits, which may reduce coughing upon contact and/or aid in the healing of the mouth and/or throat.

Personal healthcare compositions may contain diluents. Non-limiting examples of diluents may include microcrystalline cellulose, silanized microcrystalline cellulose such as SMCC 90 (commercially available from JRS Pharma, Patterson, NY, USA), dextrose, mannitol, sorbitol, maltodextrin, maltitol, and combinations thereof. Suitable diluent levels are about 20% to about 90% by weight of the composition, or about 30% to about 85%, or about 40% to about 83%, or about 50% to about 80%, or about 60% to about 78%.

Personal healthcare compositions may contain disintegrants. Disintegrants may be included to provide rapid disintegration of a solid oral dosage form upon administration. Non-limiting examples of disintegrants may include crospovidone, sodium carboxymethyl starch, sodium crospovidone carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, guar gum, sodium alginate, and mixtures thereof. Suitable disintegrant levels are from about 1% to about 20% by weight of the composition, or from about 2% to about 15%, or from about 3% to about 10%, or from about 5% to about 8%.

On one hand, the composition may contain mannitol and crospovidone to provide rapid disintegration and dissolution. One advantage of using a soluble sugar such as mannitol is that it absorbs water and dissolves rapidly. One advantage of using a disintegrant such as crospovidone is that it absorbs water and swells, leading to the breakdown of the dosage form. When the dosage form breaks down, it is exposed to liquids, such as saliva in the mouth, and dissolves even more quickly. The ratio of mannitol to crospovidone may be about 15:1, alternatively about 13:1, or alternatively about 10:1.

Personal healthcare compositions may contain lubricants. Non-limiting examples of lubricants may include sodium stearate fumarate, magnesium stearate, calcium stearate, zinc stearate, stearic acid, glyceryl behenate, hydrogenated vegetable oil, talc, polyethylene glycol, mineral oil, and combinations thereof. Suitable levels of the lubricant are from about 0.05% to about 5% by weight of the composition, or from about 0.1% to about 3%, or from about 0.25% to about 1.5%, or from about 0.3% to about 1%, or from about 0.4% to about 0.6%.

On one hand, personal healthcare compositions can be non-Newtonian or thixotropic fluids, exhibiting a reduced apparent viscosity when subjected to shear forces but a high apparent viscosity at rest. One advantage of non-Newtonian fluids is that they allow application by spraying immediately after the application of shear forces (such as those generated by vigorous shaking) using a pump-jet device or a squeeze-jet can, while keeping the sprayed material relatively immobile, at least temporarily, on mucous membranes or skin. Preferably, the composition can have a very rapid viscosity recovery rate after the shear force is withdrawn.

Personal healthcare compositions may contain rheology modifiers. Non-limiting examples of rheology modifiers may include sodium carboxymethyl cellulose, alginate, carrageenan (including I, K, and L carrageenan and combinations thereof), carbomer, galactomannan, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl chitosan, sodium carboxymethyl dextran, sodium carboxymethyl starch, microcrystalline cellulose, mixtures of microcrystalline cellulose and sodium carboxymethyl cellulose (commercially available from FMC Corporation, Philadelphia, PA under RC-591), xanthan gum, and combinations thereof. Suitable levels of the rheology modifier may be from about 0.5% to about 15%, or from about 1% to about 12%, or from about 2% to about 6%, all percentages being by weight of the composition. Rheology modifiers not only provide viscosity benefits but also allow for longer application to the nose and throat to soothe and/or deliver selected medications.

Personal healthcare compositions may also contain humectants. These humectants may be hygroscopic substances, such as glycerin, polyethylene or other glycols, polysaccharides, aloe vera, etc., used to inhibit moisture loss from the composition and to increase moisturizing properties.

Personal healthcare compositions may contain acidifying agents. Acidifying agents may include organic acids, pyroglutamic acid, and combinations thereof. Suitable organic acids may include, but are not limited to, ascorbic acid, monocarboxylic acids, dicarboxylic acids, tricarboxylic acids, and mixtures thereof. Specific, non-limiting examples of suitable monocarboxylic acids, dicarboxylic acids, or tricarboxylic acids include salicylic acid, fumaric acid, benzoic acid, glutaric acid, lactic acid, citric acid, malonic acid, acetic acid, glycolic acid, malic acid, adipic acid, succinic acid, aspartic acid, phthalic acid, tartaric acid, glutamic acid, gluconic acid, and mixtures thereof. Without being theoretically limited, it is believed that incorporating acids into nasal compositions can create a hostile environment for viruses without significantly irritating specific areas of the respiratory tract, such as nasal tissue. The composition may contain about 0.01% to about 10%, or about 0.05% to about 5%, or about 0.10% to about 2.5% of organic acids, all percentages being by weight of the composition.

Personal healthcare compositions may contain surfactant diffusion aids, such as polyoxyethylene (20) dehydrated sorbitan monooleate commercially available as polysorbate 80, polyoxyethylene (20) dehydrated sorbitan monolaurate commercially available as polysorbate 20, polyethylene glycol 400 stearate, polyethylene glycol, polyethylene-polypropylene glycol commercially available as poloxamer 407, and combinations thereof. The surfactant may be included in the composition at a concentration ranging from about 0.001% to about 10%, or from about 0.01% to about 5%, or from about 0.1% to about 3% by weight of the composition.

Additional components

The personal healthcare compositions described herein may optionally contain one or more additional components known for use in personal healthcare products, provided that the additional components are physically and chemically compatible with the components described herein, or do not otherwise unduly impair the stability, aesthetics, or performance of the product. Optional components applicable herein include substances such as preservatives, pH adjusters, chelating agents, metal compounds, pharmaceutically active ingredients, vitamins, herbal ingredients, sweeteners, sensory agents, flavoring agents, natural honey, volatile oils, aromatic components (such as camphor, eucalyptol, menthol, fragrances, etc.), antioxidants, amino acids, energy-enhancing ingredients, sleep aids, sodium chloride, and combinations thereof. Optional components may be included in the personal healthcare compositions at concentrations ranging from about 0.001% to about 20%, or from about 0.01% to about 10%, or from about 0.1% to about 5%, all percentages being by weight of the composition.

In one aspect, the personal healthcare composition may contain a preservative. Optionally, a preservative may be included to prevent microbial contamination. Non-limiting examples of preservatives may include benzalkonium chloride, chlorhexidine gluconate, phenethyl alcohol, phenoxyethanol, benzyl alcohol, sorbic acid, thimerosal, phenylmercuric acetate, methylparaben, propylparaben, butylparaben, chlorobutanol, and mixtures thereof.

In one aspect, the personal healthcare composition may contain a pH adjuster. Non-limiting examples of pH adjusters may include sodium bicarbonate, sodium phosphate, sodium hydroxide, ammonium hydroxide, sodium stannate, triethanolamine, sodium citrate, disodium succinate, and mixtures thereof. Optional pH adjusters may be included in the composition to adjust the pH to a value of about 2 to about 8, or about 2 to about 5. If present, the pH adjuster is typically included at a concentration ranging from about 0.01% to about 5.0% by weight of the composition.

In one aspect, the personal healthcare composition may contain a chelating agent. Non-limiting examples of suitable optional chelating agents may include phytic acid, disodium and calcium salts of ethylenediaminetetraacetic acid (EDTA), tetrasodium EDTA, sodium hexametaphosphate (SHMP), bis(hydroxyethyl)glycine, 8-hydroxyquinoline, and mixtures thereof. The chelating agent may be included at a concentration ranging from about 0.001% to 10% by weight of the composition, preferably from about 0.005% to about 5%, more preferably from about 0.01% to about 2%.

Personal healthcare compositions may contain metal compounds. Metal compounds suitable for use herein include those containing metal ions selected from the group consisting of: manganese (Mn), silver (Ag), zinc (Zn), tin (Sn), iron (Fe), copper (Cu), aluminum (Al), nickel (Ni), cobalt (Co), and mixtures thereof. Non-limiting examples of metal compounds suitable for use herein include zinc acetate, zinc chloride, zinc ascorbate, zinc gluconate, zinc pyroglutamate, zinc succinate, zinc sulfate, zinc edetate, and mixtures thereof. Zinc acetate is the most preferred metal compound.

When personal healthcare compositions contain metal compounds containing zinc ions, the zinc ions are believed to provide antiviral properties. Zinc ions have been shown to be antiviral and antibacterial. They are believed to inhibit the cleavage of rhinovirus peptides, preventing the replication and formation of infectious viral particles. Zinc ions reduce the ability of rhinovirus to penetrate cell membranes, partly by decreasing the expression of the intercellular adhesion molecule ICAM. Zinc ions have also been shown to stimulate the production of T-cell lymphocytes, including natural antivirals and interferon-γ. These zinc ions stabilize the cell membrane, protect cells from cytotoxic agents, and prevent cell leakage. In addition, metal ions such as iron, silver, copper, and zinc are known to provide antiviral properties for the prevention and treatment of cold and flu-like symptoms. The concentration of the metal compound in the personal healthcare composition may range from about 0.001% to about 20%, or from about 0.01% to about 10%, or from about 0.05% to about 5%, or from about 0.1% to about 2%, or from 0.2% to about 1%, all percentages being by weight of the composition.

Non-limiting examples of active pharmaceutical ingredients may include menthol; anesthetics such as benzocaine and lidocaine; decongestants such as phenylephrine, pseudoephedrine, xylometazoline, and oxymetazoline; antihistamines such as doxylamine, diphenhydramine, loratadine, and cetirizine; and expectorants such as guaifenesin and ambroxol. Ambroxol and bromhexine; analgesics such as acetaminophen (APAP), ibuprofen, ketoprofen, diclofenac, naproxen, and aspirin; antitussives such as dextromethorphan, codeine, chlophedianol, and levodropropizine; their free salts and addition salts; and combinations thereof. The pharmaceutically active ingredient may be present at levels of about 0.01% to about 25%, or about 0.05% to about 15%, or about 0.1% to about 10%, or about 1% to about 5%, all percentages being by weight of the composition. In one aspect, the personal healthcare composition may comprise at least one aptamer disclosed herein and one or more pharmaceutically active ingredients to relieve one or more symptoms and inhibit rhinovirus binding.

Non-limiting examples of vitamins may include vitamin A, vitamin C, vitamin D2, vitamin D3, vitamin E, vitamin K1, vitamin K3, vitamin B1, vitamin B3, folic acid, vitamin B12, vitamin B3, vitamin B7, and combinations thereof. In some aspects, the composition may contain from about 0.1% to about 10%, alternatively from about 1% to about 8%, or alternatively from about 2% to about 6% of the vitamins by weight of the composition.

Non-limiting examples of herbal ingredients may include rosemary (leaf), ginger, lemon balm, green tea, holy basil, oregano, thyme, ginseng, false purslane, chamomile, valerian, rosemary, turmeric, grape seed, blueberry, coffee, curcumin, elderberry, medicinal marshmallow root, ivy leaf, black tea, white tea, oolong tea, green tea, and combinations thereof. In some aspects, the herbal ingredient may be a whole herb or plant part, extract, powder, concentrate, or combination thereof. In some aspects, the composition may contain from about 0.1% to about 10% by weight of the composition, alternatively from about 1% to about 8%, or alternatively from about 2% to about 6%.

On the one hand, sweeteners can be selected from the group consisting of sugar alcohols, synthetic sweeteners, high-intensity natural sweeteners, and combinations thereof.

Non-limiting examples of nutritional sweeteners may include sucrose, dextrose, glucose, fructose, lactose, tagatose, maltose, trehalose, high-fructose corn syrup, and combinations thereof. The nutritional sweetener may be present in an amount of about 1% to about 99% by weight of the composition, or about 4% to about 95%, or about 10% to about 70%, or about 15% to about 60%, or about 25% to about 50%, or in another example, about 35% to about 45%.

Non-limiting examples of sugar alcohols may include xylitol, sorbitol, mannitol, maltitol, lactitol, isomaltitol, erythritol, and combinations thereof. The sugar alcohol may be present in amounts of about 5% to about 70%, about 10% to about 60%, about 15% to about 55%, about 25% to about 50%, or about 30% to about 45% by weight of the composition.

Non-limiting examples of synthetic sweeteners may include aspartame, potassium butyrate, alitane, sodium saccharin, sucralose, neotame, cyclosulfonates, and combinations thereof. The synthetic sweetener may be present in an amount of about 0.01% to about 10%, about 0.05% to about 5%, about 0.1% to about 3%, about 0.2% to about 1%, or about 0.1% to about 0.5% by weight of the composition.

Non-limiting examples of high-intensity natural sweeteners may include neohesperidin dihydrochalcone, hedyoside, rebaudioside A, rebaudioside C, dulcitin, monoammonium glycyrrhizinate, arbutin, and combinations thereof. The high-intensity natural sweetener may be present in an amount of about 0.01% to about 10%, or about 0.05% to about 5%, or about 0.1% to about 3%, or about 0.5% to about 1% by weight of the composition.

Personal healthcare compositions may include flavoring systems, which may include sensory agents, flavoring agents, salivating agents, and combinations thereof.

Personal healthcare compositions may contain sensory agents. Non-limiting examples of sensory agents may include cooling sensory agents, warming sensory agents, tingling sensory agents, and combinations thereof. Sensory agents may deliver sensory signals to the oral cavity, pharynx, nasal cavity, and/or sinus passages, allowing the personal healthcare composition to be perceived by the user as immediately effective in relieving ailments and/or providing a soothing sensation.

Non-limiting examples of cooling agents may include WS-23 (2-isopropyl-N,2,3-trimethylbutyramide), WS-3 (N-ethyl-p-menthane-3-carboxamide), WS-30 (1-glycero-p-menthane-3-carboxylate), WS-4 (ethylene glycol-p-methane-3-carboxylate), WS-14 (N-tert-butyl-p-menthane-3-carboxamide), WS-12 (N-(4-,ethoxyphenyl)-p-menthane-3-carboxamide), WS-5 (ethyl 3-(p-menthane-3-carboxamido)acetate), menthol, levomenthol, l-menthone glyceryl ketal (sold by Symrise, Holzminden, Germany in MGA), (-)-menthyl lactate (sold by Symrise, Holzminden, Germany in ML), (-)-menthoxypropane-1,2-diol (sold by Vantage Specialty) Ingredients, Inc., Warren, NJ (sold for 10), 3-(l-menthoxy)-2-methylpropane-1,2-diol, (-)-isomenthyl alcohol (sold by Takasago International, Tokyo, Japan under Coolact), cis & trans-p-menthane-3,8-diol (sold by Takasago International under 38D), pyrrolidone carboxylic acid menthyl ester (sold by Givaudan Active) (available at Beauty, Verbuer, Switzerland) (lR,3R,4S)-3-mentheptyl-3,6-dioxaneheptaate (available at Firmenich, Geneva, Switzerland), (lR,2S,5R)-3-mentheptylmethoxyacetate (available at Firmenich), (lR,2S,5R)-3-mentheptyl-3,6,9-trioxanedecanoate (available at Firmenich), (lR,2S,5R)-mentheptyl-11-hydroxy-3,6,9-trioxanedecanoate (available at Beauty, Verbuer, Switzerland) Purchased from Firmenich), (lR,2S,5R)-3-menthyl(2-hydroxyethoxy)acetate (available from Firmenich), Icilin also known as AG-3-5 (chemical name l-(2-hydroxyphenyl)-4-(3-nitrophenyl)-3,6-dihydropyrimidin-2-one), 4-methyl-3-(l-pyrrolidinyl)-2[5H]-furanone, peppermint oil, spearmint oil, L-menthyl succinate, L-menthyl glutarate, 2-1-menthoxyethanol (5), 3-1-menthoxypropane-1,2-diol (sold by Takasago International at TK10), N-(4-cyanomethylphenyl)-p-menthylcarboxamide (sold by Givaudan at Evercool ^TM 180), and combinations thereof. The cooling sensation agent may be present in about 0.001% to about 1%, or about 0.01% to about 0.5%, or about 0.02% to about 0.25%, or about 0.03% to about 0.10% by weight of the composition.

Non-limiting examples of thermosensitive agents may include vanillyl alcohol n-butyl ether (sold by Takasago International as TK-1000), Heatenol™ (available from Sensient Pharmaceutical, St. Louis, MO), Optaheat (sold by Symrise, Holzminden, Germany), ginger extract, capsicum tincture, cinnamon, capsaicin, curry, vanillyl isobutyrate, nonivamide, vanillyl butyl ether (commercially available from VBE), piperine, and combinations thereof. The thermosensitive agent may be present in amounts from about 0.005% to about 2%, or from about 0.01% to about 1%, or from about 0.1% to about 0.5% by weight of the composition.

Non-limiting examples of flavorings may include natural flavorings, artificial flavorings, artificial extracts, natural extracts, and combinations thereof. Non-limiting examples of flavorings may include vanilla, honey, lemon, lemon honey, cherry vanilla, peach, honey ginger, chamomile, cherry, cherry frosting, mint, vanilla mint, dark berry, blackberry, raspberry, peppermint, spearmint, peach, acai berry, cranberry, honey cranberry, tropical fruit, dragon fruit, goji berry, red-stemmed mint, pomegranate, blackcurrant, strawberry, lemon, lime, peach ginger, orange, orange peel frosting, apricot, anise, ginger, jackfruit, star fruit, blueberry, mixed fruit punch, lemongrass, banana, strawberry-banana juice, grape, raspberry, lemon-lime juice, winter green mint, chewing gum, sour honey lemon, green apple, apple, tangerine, grapefruit, kiwi, pear, orange, tangerine lime, menthol, and combinations thereof. The flavoring agent may be present in about 0.05% to about 10% by weight of the composition, or about 0.1% to about 8%, or about 0.2% to about 6%, or about 0.4% to about 3%, or about 0.6% to about 1.5%.

This document also describes a kit comprising the personal healthcare composition described herein. In one aspect, the kit may include a delivery device and a personal healthcare composition contained within the delivery device. In another aspect, the kit may optionally include at least one additional component, such as a supplement or vitamin composition.

This document also describes methods for providing one or more health benefits, comprising administering to a subject in need a personal healthcare composition comprising at least one aptamer disclosed herein, as described herein, wherein the aptamer has binding affinity for ICAM-1. Non-limiting examples of one or more health benefits may include providing a physical barrier to prevent rhinovirus binding and entry into cells, thereby helping to prevent the formation of colds caused by rhinovirus, reducing the severity and/or duration of colds caused by rhinovirus, reducing the likelihood of catching a cold, and combinations thereof.

Example

The following examples illustrate non-limiting embodiments of the invention described herein. Exemplary personal healthcare compositions can be prepared using conventional formulation and mixing techniques. It should be understood that those skilled in the art can make other modifications to these personal healthcare compositions without departing from the spirit and scope of the invention. The following are non-limiting examples of the personal healthcare compositions described herein.

Oral Composition Examples

throat spray

Oral dissolving tablet formulation

Liquid composition

throat lozenge composition

Nasal Composition

Saline nasal spray composition

Nasal spray composition

Additional nasal spray composition

VI. Examples

Example 1. Aptamer selection and next-generation sequence characterization .

A. Choosing a strategy

One object of this invention is to develop aptamers that not only specifically bind to the ICAM-1 receptor but also do so in a manner that inhibits the binding of viral particles to the receptor protein. The selection of aptamers targeting only the extracellular domain of the ICAM-1 receptor may not be sufficient to inhibit viral binding to the same protein because the aptamers are relatively small and their inhibitory footprint will be limited to the epitope they bind to. If the epitope to which the aptamer binds does not involve viral binding to the ICAM-1 receptor, then they will not inhibit viral particle binding.

This objective was consciously incorporated into the selection strategy, firstly through several rounds of positive selection targeting the extracellular domain of the ICAM-1 protein (SEQ ID NO: 214); secondly, by applying double positive selection, aptamers were enriched against the background of nasal cells for binding to the extracellular domain of ICAM-1; thirdly, by applying anti-selection against HEK293 cells carrying similar receptor proteins (ICAM-3 SEQ ID NO: 232 and ICAM-5 SEQ ID NO: 234; SEQ ID NO: 263); and fourthly, by selecting pathways based on specific desired and undesired aptamer binding outcomes, including by specifically eluting bound aptamers from nasal cells through the addition of rhinovirus particles, by inhibiting aptamer binding to ICAM-1 cells through pre-application of rhinovirus particles, positive selection targeting HEK293 cells, positive selection targeting the extracellular domain of ICAM-1, and double positive selection targeting the extracellular domains of both ICAM-1 and nasal cells.

When the ICAM-1 receptor is present on nasal cells, dual positive selection (of both ICAM-1 and the extracellular domain of the nasal cell) ensures that the enriched aptamers are conducive to binding to the ICAM-1 receptor. If selection is performed only on the extracellular domain of ICAM-1, epitopes that are not present in vivo may be present. If selection is performed only on nasal cells, the aptamers will be enriched for binding targets other than ICAM-1 on the cell surface.

Anti-selection was performed on HEK293 cells to drive the enrichment of aptamers that bind to the N-terminus of the ICAM-1 extracellular domain. HEK293 cells express other members of the ICAM receptor family, ICAM-3 and ICAM-5. These receptor proteins differ from ICAM-1 primarily at their N-terminus in their extracellular domain. The N-terminus of the ICAM-1 receptor is the region of the extracellular domain that binds to rhinovirus particles. Therefore, this anti-selection step was included to drive aptamer selection toward those aptamers that would inhibit rhinovirus binding to nasal cells.

Finally, once the aptamer library was enriched by double positive selection targeting the extracellular domains of ICAM-1 and nasal cells and anti-selection targeting HEK293 cells, the enriched library was separated into aliquots and applied to several different targets, including persistent double positive selection, positive selection targeting HEK293 cells, positive selection targeting the extracellular domains only, selection based on elution of rhinovirus particles from aptamers that bind to nasal cells, and selection based on inhibiting aptamer binding to nasal cells by pretreatment with rhinovirus particles.

Each of these selected libraries was characterized by next-generation sequencing. Aptamers exhibiting higher enrichment for any of the double-positive selection, extracellular domain selection, and rhinovirus particle initiation selection methods, and lower enrichment specifically for HEK293, are the desired sequences for inhibiting rhinovirus binding to nasal cells. These aptamers are used as parental aptamers, and the aptamers disclosed herein are truncated from the parental aptamers.

B. Human cell growth

B.1. Growth conditions of human nasal epithelial cells

Primary human nasal epithelial cells (HNEpC; PromoCell, catalog number C-21060) were grown in airway epithelial cell growth medium (PromoCell, catalog number C-21160) at 37°C and 5% _CO2 .

B.2. Growth of HEK293 cells

HEK293 cells purchased from ATCC (CRL-1573) were grown in Eagle's minimum essential medium (EMEM) + 10% fetal bovine serum (FBS) at 37°C and 5% _CO2 .

B.3. Human rhinovirus A16 suspension

Purchase UV-inactivated HRV16 virus particles (Zeptometrix Corporation) and store at -80°C until use. Calculate the concentration of virus particles (VP) to be 98,700 vp/mL.

C. Parent aptamer selection

C.1. Library Preparation

In the first step, a DNA library of approximately ^10-15 different sequences (TriLink BioTechnologies), containing a random region of 40 nucleotides flanked by two conserved regions, a forward primer recognition sequence (5'-GGGTGCATCGTTTACGC-3'; SEQ ID No 224) and a 3' reverse primer recognition sequence (5'-CTGCTGCTGAGGAAGGATATGAG-3' SEQ ID No 225) (see Figure 1), was transcribed into RNA using a mixture of 2'-fluoropyrimidine nucleotides (2F-UTP and 2F-CTP) and native purine nucleotides.

In summary, approximately 1.66 nanomolars of single-stranded DNA were amplified in a 390 x 50 μL PCR reaction using primers Lib7_T7 Fwd (sequence: 5'-TAATACGACTCACTATA G GGTGCATCGTTTACGC-3' (SEQ ID No 226), transcription begins with the first underlined G) and Lib7_T7 Rvs (sequence: 5'-CTCATATCCTTCCTCAGCAGCAG-3' (SEQ ID No 227) for 4 cycles. The amplified DNA was purified using a Genejet PCR purification kit (Fisher Scientific, catalog number K0701). This amplification of the ssDNA library produced a dsDNA library with the T7 promoter, which was used as a template to generate an RNA library for selective modification.

Following DNA amplification, 52 μg of purified dsDNA was transcribed in a 26 x 20 μL transcription reaction using a mutant T7 polymerase (T7 R&D polymerase, Lucigen, catalog number D7P9205K) and a mixture of rATP, rGTP, and modified nucleotides 2F-UTP and 2F-CTP. NTPs were mixed at a 3:1 modified to unmodified ratio. Each reaction mixture contained 4 μL 5x T7 R&D polymerase, 1 μL of the 3:1 NTP mixture, 2 μL DTT (0.1 M), 0.7 μL T7 R&D polymerase, 1.2 μL of inorganic pyrophosphatase, 0.5 μL of RNase inhibitor, and 10.6 μL of DNA template. The reaction was incubated at 37°C for 16 hours.

The transcription library was DNase-treated using a reaction mixture consisting of 10 μL 10x DNase buffer, 4 μL DNase I, 66 μL RNase-free water, and 20 μL of transcription reaction solution. The reaction mixture was then incubated at 37°C for 30 minutes, followed by the addition of 1 μL 0.5M EDTA and mixing. The mixture was then further incubated at 75°C for 10 minutes and purified using the Monarch RNA Cleansing Kit (New England Biolabs, catalog number T2040L).

C.2. Fix ICAM-1 onto His-PurNi-NTA resin

Lyophilized ICAM-1 protein (50 μg Ray-Biotech, catalog number: 228-21751-2) with a His tag at the C-terminus was resuspended in 100 μL of _sH₂O (final concentration 0.5 μg/μL or 9.88 μM). The solution was aliquoted and stored at -20°C until use. The protein sequence is: QTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGD QRLNPTVTYGNDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSAT LEVAGQLIHKNQTRELRVLYGPRLDERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEVDHHHHHH(SEQ ID No. 228).

Transfer aliquots of His-Pur Ni-NTA (Fisher Scientific, catalog number PI88221) resin to 0.6 mL tubes and centrifuge at 700 x g for 2 min. Remove the supernatant and wash the resin three times with 500 μL PBS buffer (pH 7.4). Then, incubate aliquots of ICAM-1 protein in 1xPBS buffer (pH 7.4) with His-Pur Ni-NTA resin overnight at 4 °C, mixing simultaneously. For Select Round 1, immobilize 300 picomoles of ICAM-1 protein onto 50 μL of resin. For subsequent rounds, immobilize 50 picomoles of ICAM-1 protein onto 25 μL of resin. After protein immobilization, transfer the resin to a 1 mL tube fitted with a glass frit filter and wash with 2 mL of 1xPBS buffer. Finally, add an aliquot of 0.5 mM–1 mM imidazole in 1x PBS buffer and incubate with the resin at 4°C for 30 minutes to block unreacted binding sites on the resin. Wash the resin three times with 1 mL of the aliquot in 1x PBS buffer.

To perform negative selection with imidazole-capped resin, aliquots of His-Pur Ni-NTA resin were incubated with an appropriate concentration of imidazole in 1x PBS buffer for 30 minutes to block unreacted binding sites on the resin, followed by washing with 1x selection buffer. The selection buffer used in all embodiments of this application was Dulbecco's PBS buffer supplemented with calcium chloride (CaCl2, 0.9 mM), magnesium chloride (MgCl2, 0.49 mM), potassium chloride (KCl, 2.67 mM), potassium dihydrogen phosphate (KH2PO4, 1.47 mM), sodium chloride (NaCl, 137.93 mM), and disodium hydrogen phosphate (Na2HPO4, 8.06 mM).

C.3. Overview of Parental Aptamer Selection

Aptamer selection was performed in fourteen selection rounds (“SR”), as shown in Figure 2. Selection rounds 1 through 5 enriched the aptamer library with sequences binding to ICAM-1 immobilized on Ni-NTA resin. In selection rounds 6 through 9, the aptamer library underwent the same ICAM-1 immobilization Ni-NTA resin procedure, and the eluted aptamers were further enriched toward sequences binding to human nasal epithelial cells (HNEpC), a process referred to as double positive selection. In selection rounds 10 and 11, anti-selection was performed against HEK293 cells and positive selection against HNEpC. Selection rounds 12 through 14, shown in Figure 3, broke through to different selection conditions and were referred to as cleavage. Five different cleavages were performed: cleavage A: nasal epithelial cells, cleavage B: HEK293 cells, cleavage C: ICAM-1 protein, cleavage D: human rhinovirus A16 (HRV16) elution, and cleavage E: HRV16 blocking.

C.4. Parental aptamer selection process

C.4.1 Selecting Round 1

Aptamer selection round 1 was completed by positive selection against Ni-NTA resin immobilized with ICAM-1. The RNA library (generated as described in Section C.1) was heated to 45°C for 10 minutes and then cooled to room temperature for 10 minutes. The prepared aptamer library was then added to 300 picomoles of ICAM-1 (prepared as described in Section C.2) immobilized on Ni-NTA resin and incubated by rotation at room temperature for 30 minutes. Unbound RNA was washed off the resin with 500 μL of selection buffer (pH 7.4).

The bound RNA was then eluted twice by adding an aliquot of 200 μL of 6M urea to the resin and incubating the suspension at 85°C for 5 minutes. The recovered RNA library was collected and purified using the Monarch RNA Removal Kit.

The collected aptamer library is reverse transcribed according to the protocol specified by the Protoscript II reverse transcriptase manufacturer. The number of reverse transcription reactions varies depending on the amount of RNA entering that particular selection round.

Then, the reverse-transcribed aptamer library was amplified by polymerase chain reaction (PCR) using a standard PCR protocol and the following amplification steps:

Step 1: 95℃ for 5 minutes

Step 2: 95℃ - 10 seconds

Step 3: 56℃ - 15 seconds

Step 4: 72℃ - 30 seconds

Repeat steps 2 through 4 for 4 cycles.

Step 5: 95℃ - 10 seconds

Step 6: 59℃ - 15 seconds

Step 7: 72℃ - 30 seconds

Repeat steps 5 through 7 for up to 26 cycles.

The PCR-amplified dsDNA aptamer library was then transcribed back into RNA and treated with DNase using the protocol described in Section C.1.

C.4.2 Select rounds 2 to 5

Rounds 2 through 5 incorporate two selection strategies: negative selection for imidazole-capped Ni-NTA resin and positive selection for Ni-NTA resin immobilized with ICAM-1 (see Figure 2). Negative selection is performed to select aptamer sequences that do not bind to imidazole-capped Ni-NTA resin (prepared as described in Section C.2). First, an aliquot of 50 μL of imidazole-capped resin is transferred to a 1 mL cartridge containing 20 μm glass frit and washed twice with 1 mL of selection buffer for the aliquot. Then, the RNA library prepared from the previous selection round is heated to 45°C for 10 minutes and cooled to room temperature for 10 minutes. The RNA library is added to the cartridge and incubated with the imidazole-capped Ni-NTA resin at room temperature for 30 minutes. After incubation, the flow-through solution is collected. The cartridge is then washed with an aliquot of 500 μL of selection buffer, and the solution is collected. The flow-through solution and column wash collection are pooled together and purified using the Monarch RNA Removal Kit according to the manufacturing protocol.

The RNA library obtained from negative selection was then positively selected for sequences that bound to Ni-NTA resin immobilized in ICAM-1 (prepared as described in Section C.2). Briefly, the RNA library was heated to 45°C for 10 minutes and then cooled to room temperature for 10 minutes. The RNA library was then added to 50 picomoles of ICAM-1 immobilized on Ni-NTA resin (prepared as described in Section C.2) and incubated by rotation at room temperature for 30 minutes. Unbound RNA was washed off the resin with an aliquot of 500 μL of selection buffer. The number of washes varied depending on the selection rounds and the number of positive selections completed, and was predetermined by selection modeling. The bound RNA library was then eluted twice by adding an aliquot of 200 μL of 6M urea to the resin and incubating the suspension at 85°C for 5 minutes. The eluted RNA library was collected and purified using the Monarch RNA Removal Kit, followed by reverse transcription, PCR amplification, transcription, and DNase treatment as described in Sections C.1 and C.4.1.

C.4.3. Select rounds 6 to 9.

The RNA aptamer libraries enriched in selection rounds 1 through 5 were further enriched in selection rounds 6 through 9, employing two selection strategies: positive selection using Ni-NTA resin fixed with ICAM-1 and another positive selection targeting human nasal epithelial cells (HNEpC) expressing the ICAM-1 receptor. This set of selection rounds is referred to as “double positive selection.” In selection round 8, two positive selections targeting HNEpC were performed (i.e., “triple positive selection”).

In selection rounds 6 and 7, the RNA library was resuspended in 500 μL of 1x selection buffer. The first positive selection (for ICAM-1-fixed Ni-NTA resin) was initiated by adding the resuspended RNA to ICAM-1 fixed on Ni-NTA resin, followed by incubation at 37°C for 30 min. Unbound RNA was discarded, and the resin was washed with an aliquot of 500 μL of 1x selection buffer. For the elution step, an aliquot of 200 μL of 6M urea was added to the resin and incubated at 85°C for 5 min, and the eluent was collected. The elution step was repeated, and the eluents were pooled and washed using the Monarch RNA Purification Kit.

The second positive selection began by preparing HNEpC cells by aspirating culture medium (approximately 3 mL) from a 6-well plate used for cell growth, followed by washing the cells three times with 3 mL of pre-warmed 1x selection buffer. Immediately, 1 mL of RNA library in 1x selection buffer was added to the washed cells, and the cells were incubated at 37°C and 50 rpm for 30 minutes. After 30 minutes of incubation, the supernatant containing approximately 50% cells was collected, and the cells were pelleted at 500 x g for 2 minutes and washed twice with 200 μL of pre-warmed 1x selection buffer. The cell pellet was collected, and bound RNA was eluted from the cells by adding 6 M urea, followed by incubation at 85°C for RNA purification.

The adhered cells (approximately 50% of the cells remaining) were washed twice with 1 mL of pre-warmed 1x selection buffer. Then, an aliquot of 1 mL of 10 mM EDTA was added and incubated with the cells at 37°C for 15 min at 50 rpm. The EDTA-treated cells were precipitated at 500 x g for 2 min. Then, an aliquot of 200 μL of 6 M urea was added to the precipitate, and the suspension was heated to 85°C for 5 min, followed by centrifugation at 13,000 rpm to recover the RNA aptamers from the supernatant. The elution steps were repeated, the eluates were combined, and the RNA aptamers were purified. The purified sample was subjected to reverse transcription, PCR amplification, and transcription according to the protocols in Sections C.1 and C.4.1.

In selection rounds 8 and 9, EDTA boosting of cells was removed from the protocol, and cell-bound RNA was eluted using 6M urea while still attached to the 6-well plate. Additionally, a negative selection step was included in both rounds to remove any RNA sequences bound to the plastic of the 6-well plate. For negative selection, the RNA library was resuspended in 1 mL of 1x selection buffer and then heated to 37°C for at least 10 minutes. One well of the 6-well plate was pre-washed twice with 1 mL of 1x selection buffer. The heated RNA library was then added to the well and incubated at 37°C and 50 rpm for 30 minutes. The solution in the well was collected and brought to a volume of 1 mL using selection buffer. The resulting 1 mL RNA library solution was incubated with HNEpC grown in the 6-well plate at 37°C and 50 rpm for 1 hour. Unbound RNA was removed from the cells, and the cells were washed twice with 1 mL of 1x selection buffer (preheated to 37°C). Bound RNA was eluted by adding 1 mL of 6M urea and incubating the cells at 85°C for 5 minutes. Repeat the elution steps. Collect the eluents together and purify the RNA using the Monarch RNA Purification Kit. As previously described, the selected RNA was subjected to reverse transcription, PCR amplification, transcription, and DNase treatment.

C.4.4. Select rounds 10 and 11

In selection rounds 10 and 11, negative selection was introduced for HEK293 cells (see Figure 2). HEK293 cells do not express the ICAM-1 receptor, which allows for anti-selection against sequences that bind elsewhere on the cell surface that are not ICAM-1.

HEK293 cells were grown in 6-well plates and used at 80% or higher confluence. Cells were prepared by removing and discarding all culture medium from the wells and washing the cells three times with 3 mL of pre-warmed 1x selection buffer. The prepared RNA library was then added to the cells, and the library and cell solution were incubated at 37°C for 1 hour with gentle shaking (50 rpm). After incubation, the supernatant containing unbound RNA was removed and collected. The cells were then washed with 1 mL of pre-warmed 1x selection buffer, and the solution was also collected. The collected RNA solutions were combined and purified using the Monarch RNA Removal Kit. The purified RNA library was then subjected to positive selection rounds against HNEpC according to the same protocol as described in selection rounds 8 and 9 (see Section C.4.3). Two positive selections were performed in selection round 10, and a single positive selection was performed in selection round 11.

C.4.5. Selecting rounds 12 to 14: Nasal epithelial cell division

During nasal epithelial cell divisions in selection rounds 12 to 14 (see Figure 3), the RNA library collected from selection round 11 was further negatively selected for HEK293 cells, followed by positive selection with HNepC using the protocol described in Section C.4.4.

C.4.6. Select rounds 12 to 14: HEK293 cell division

During HEK293 cell divisions in selection rounds 12 through 14 (see Figure 3), the RNA library collected in selection round 11 was enriched toward sequences binding to HEK293 cells. The protocol for this selection round followed the procedure for selection rounds 10 through 11 as described in Section C.4.4, excluding selection with HNepC.

C.4.7. Select rounds 12 to 14: ICAM-1 protein cleavage

During ICAM-1 splits in selection rounds 12 through 14 (see Figure 3), the RNA library collected in selection round 11 is enriched toward sequences bound to ICAM-1 immobilized on Ni-NTA resin. The scheme for this selection round follows the procedure for selection round 1 described in Sections C.1 and C.4.1.

C.4.8. Select rounds 12 to 13: Human rhinovirus A16 (HRV16) elution and division

HRV16 elution occurred only during selection rounds 12 and 13 (see Figure 3). The RNA library collected in selection round 11 was further enriched by negative selection against HEK293 cells, followed by positive selection against HNepC using human rhinovirus A16 (HRV16) particles to elute the aptamer library. Negative selection against HEK293 cells followed the same protocol as selection rounds 10 and 11 described in Section C.4.4, but without the selection against HNepC.

After negative selection with HEK293 cells, the collected RNA was diluted in 1x selection buffer and heated to 37°C for 15 minutes. HNepC cells were washed three times with 1 mL of pre-warmed selection buffer, and the heated RNA library was added to the cells and incubated at 37°C and 50 rpm for 1 hour. After incubation, unbound RNA was removed and discarded. The recovered cells were washed ten times with 1 mL of pre-warmed 1x selection buffer. Then, a suspension of 50% (v/v) viral particles (VP) (see Section B.3) in 1x selection buffer was mixed with the cells and incubated at 37°C and 50 rpm for 1 hour. The supernatant was collected and purified and reverse transcribed into RNA according to the protocols described in Sections C.1 and C.4.1.

C.4.9. Select rounds 12 and 13: HRV16 block cell division.

HRV16 was used to block cell division during selection rounds 12 and 13 (see Figure 3). The RNA library from selection round 11 was further enriched by negative selection of HEK293 cells, followed by positive selection of HNepC with HRV16, which binds to the ICAM-1 receptor, before exposing the cells to the RNA library. Negative selection of HEK293 cells followed the same protocol as selection rounds 10 and 11 described in Section C.4.4, except for selection with HNepC.

Following negative selection of HEK293 cells, a suspension of 50% (v/v) viral particles (VP) in 1x selection buffer was prepared. The suspension was then heated to 37°C for 15 minutes and mixed with pre-washed HNepC cells, followed by incubation at 37°C and 50 rpm for 1 hour. After incubation, all unbound VPs were removed and discarded. The RNA library recovered from negative selection was then resuspended in 1x selection buffer, added to the cells, and incubated at 37°C for 1 hour. The supernatant containing unbound RNA was collected, purified, and reverse transcribed according to the protocols described in Sections C.1 and C.4.1.

D. Parental aptamer sequencing

After 14 selection rounds, the aptamer libraries were sequenced. In summary, the selection libraries from rounds 10 to 14 were prepared for next-generation sequencing (NGS) via a two-step PCR process. In the first step, distinct hex codes (6-base sequences) and a portion of a universal sequencing primer were added to the 5' end of each aptamer library. In the second step, the complete universal sequencing primer was added to both ends. Following the second PCR step, these libraries were purified by acrylamide electrophoresis and their relative quantities were equilibrated. These libraries were then pooled and sent to Sick Children's Hospital in Toronto for NGS using an Illumina HiSeq 2500 instrument.

The sequencing data were tabulated and analyzed. A total of 16,116,086 sequences were analyzed, with each library containing over 200,000 sequences. Sequences from selection round 14 (nasal epithelial cell division) were sorted by copy number and named in descending order, with the sequence having the highest copy number named Nas.R-1. These preceding sequences are listed in Table 4.

The copy number of the preceding sequences from selection round 14 was determined on libraries obtained from other selection rounds. Finally, the frequency of each sequence was calculated by dividing the observed copy number by the total number of sequences observed in a particular selection library. The enrichment trajectories of the top 20 sequences in terms of frequency across different selection rounds were plotted (see Figure 4). These sequences enriched at a similar rate during the selection period.

E: Synthesis of truncated aptamers

As disclosed in Example 2A.1 below, a suitable truncated sequence identified by the theoretical analysis shown in Example 4 below was synthesized. The synthesized truncated sequence was tested in a binding assay (Example 2A.4) to confirm its binding affinity for intercellular adhesion molecule 1 (ICAM-1) and its ability to reduce the binding of one or more human rhinoviruses to ICAM-1.

Example 2. Aptamer binding specificity

The aim is to identify aptamer sequences that specifically bind to the ICAM-1 receptor and inhibit rhinovirus infection of human nasal epithelial cells. The previous section (Example 1) detailed the procedure for selecting sequences enriched in the presence of ICAM-1. This section will highlight the procedure used to identify the sequences found in Example 1 that have the highest affinity and specificity for the ICAM-1 receptor target.

Several strategies were implemented to identify key sequences from the selection process of RNA aptamers that specifically bind to and have high affinity for human epithelial cells (HNEpC) but not to HEK293 cells that do not express the ICAM-1 target. The first protocol involved exposing HNEpC and HEK293 cells to a selection of aptamer sequences, followed by incubation, elution, and quantification of the aptamer concentration bound to each cell type. Another strategy involved visualizing and identifying fluorescently labeled RNA aptamers that bind to HNEpC but not visually to HEK293 cells. The final strategy involved immobilizing the preceding RNA aptamer sequences, then allowing extracellular domains of the ICAM-1 protein and various other proteins to flow through the aptamers and using plasmon resonance to determine binding affinity. The strategies outlined above are described in detail in the following sections.

A. Detection of specificity and affinity via qPCR

A.1. Synthesis of aptamer RNA sequences

DNA oligonucleotides corresponding to the sense and antisense sequences of the RNA aptamers and the T7 RNA polymerase promoter were purchased (Integrated DNA Technologies). Each oligonucleotide was mixed at an equimolar concentration in 10 mM Tris buffer (pH 8.3) containing 50 mM KCl and 1.5 mM MgCl2, and then incubated at 95°C for 5 minutes. The modified RNA aptamers were then synthesized by transcription from a dsDNA template, followed by DNase treatment and purification as described in Sections C.1 and C.4.1 of Example 1.

A.2. Preparation of RNA aptamers, HNepC and Hek293 cells

The modified RNA aptamer was dissolved in 1x selection buffer at a concentration of 28.2 nM. HNepC or HEK293 cells were grown in the wells of a 24-well plate at densities ranging from 70% to 75% (HNepC) or 90% to 95% (HEK293 cells) as outlined in Sections B.1 and B.2 of Example 1.

A.3. qPCR Analysis Procedure

For each sample, two 20 μL qPCR reaction mixtures were prepared using a Luna qPCR universal mastermix (New England Biolabs, catalog number M3003L), 0.2 μM of each primer (forward primer: 5'-TAATACGACTCACTATAGGGTGCATCGTTTACGC-3' (SEQ ID No 226), reverse primer: 5'-CTCATATCCTTCCTCAGCAGCAG-3' (SEQ ID No 227)) and 5 μL of cDNA sample. qPCR reactions containing known amounts of sense DNA template were also prepared. PCR reactions were performed under the following conditions:

Step 1: Maintain 95℃ for 3 minutes

Step 2: Hold at 95℃ for 15 seconds

Step 3: Maintain 56℃ for 15 seconds

Step 4: Maintain 60℃ for 30 seconds

Repeat steps 2 through 4 for 40 cycles.

The Ct value of the assay sample is compared with the Ct value of a known amount of DNA sample to determine the amount of RNA bound to the cell.

A.4. Binding assay of human nasal epithelium and HEK293 aptamer

The binding specificity and affinity of six sequences (Nas.R-1, Nas.R-2, Nas.R-4, Nas.R-5, Nas.R-7, and Nas.R-8) of the preceding parental aptamer sequences identified in the testing selection process (Example 1) for HNEpC or HEK293 cells were evaluated. RNA aptamers, HNEpC, and HEK293 cells were prepared as described in Section A.2.

The aptamers were incubated with HNEpC at 37°C and 5% CO2 for 1 hour with gentle shaking every 15 minutes. Unbound RNA was removed, and the cells were washed four times with 150 μL of 1x selection buffer preheated at 37°C. To elute the bound RNA aptamers, an aliquot of 200 μL of 6M urea was added to the cells, and the cells were incubated at 85°C for 5 minutes. The elution steps were repeated, the eluates were combined, and the RNA aptamers were purified using the Monarch RNA Purification Kit according to the manufacturer's protocol. Each RNA sample was reverse transcribed in a 20 μL M-M μL V (New England Biolabs, M0253L) reverse transcriptase reaction according to the manufacturer's protocol. The reverse transcribed sequences were quantified using qPCR analysis according to the protocol described in Section A.3. HEK293 cells were treated with the same procedure. The results are shown in Figure 5. For aptamers Nas.R-2, Nas.R-4, Nas.R-5, Nas.R-7, and Nas.R-8, the binding affinity to HNepC was higher than that to HEK293 cells.

A.5. HNEpC and RH-30 aptamer binding assay: truncated deoxyribonucleotide sequences and parental deoxyribonucleotides nucleotide sequence

The binding assays were repeated using four parental deoxyribonucleic acid aptamer sequences (Nas.D-2, Nas.D-4, Nas.D-5, and Nas.D-8) that were identified as highly effective in the binding assays (Example 2, A.4) using the aforementioned parental deoxyribonucleic acid aptamer sequences. DNA was selected for these experiments to demonstrate that both RNA and DNA example aptamers are effective in this invention. Furthermore, by changing the oligonucleotides to DNA form, the stability of the oligonucleotides was improved, and the cost was reduced. The binding specificity and affinity of the parental aptamers (SEQ ID NO:102; SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:108) for human nasal epithelial cells (HNEpC) or rhabdomyosarcoma cell line (RH-30) were tested and compared with truncated aptamer sequences (SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:245, and SEQ ID NO:246). Fluorescein-amidinium (FAM)-conjugated DNA aptamers were incubated at 500 nM for 4 hours at 37 °C and 5% _CO2 on cells cultured in 24-well plates. At specific time points (0.5 h, 1 h, 2 h, 4 h), cell culture medium was aspirated, and the wells were washed three times with 1X selection buffer. To elute and lyse the cells, 200 μL of 6 M urea was added to the wells, and the plates were incubated at 37 °C for 5 minutes. The urea-aptamer solution was transferred to a 96-well microplate reader, and absorbance was measured at 495 nm. Samples were measured in triplicate. Figures 23 to 26 show the results.

As shown in Figure 23, the truncated aptamer Nas.D-2.1a (SEQ ID NO: 242) exhibited a higher binding affinity (close to 5000 RFU) compared to the parental aptamer Nas.D-2 (SEQ ID NO: 102), which showed close to 3000 RFU on HNEpC after 0.5 hours. Binding to RH-30 cells showed the same results as with the parental oligonucleotide, indicating that the truncated aptamer had a higher binding affinity, with a higher absolute number of aptamers binding to HNEpC than those binding to RH-30 cells. A second binding peak was also observed for the truncated aptamer Nas.D-2.1a (SEQ ID NO: 242) after 2 hours.

Figure 24 shows the binding of the truncated aptamer Nas.D-4.1 (SEQ ID NO:244) to the parental aptamer Nas.D-4 (SEQ ID NO:104) on HNEpC and RH-30 cells. Maximum binding was observed after 2 hours, with the absolute number of truncated sequences binding to HNEpC cells being 2.5 times that of the parental aptamer. Binding to RH-30 cells was similar for both aptamers (truncated sequence (SEQ ID NO:244) and parental sequence (SEQ ID NO:104)) and was below 500 RFU.

Maximum binding of the truncated sequence Nas.D-5 (SEQ ID NO: 245) could be observed after 0.5 hours, with the truncated aptamer binding to HNEpC more than twice as much as the parental aptamer. Binding to RH-30 cells was almost undetectable (Figure 25).

The binding characteristics of Nas.D-8.1 (SEQ ID NO:246) to HNEpC again showed two peaks with maximum binding at 0, 5, and 2 hours. The absolute number of binding oligonucleotides of the truncated aptamer (SEQ ID NO:246) was again higher than that of the parental sequence (SEQ ID NO:108). For both aptamers, binding to RH-30 cells was poorer, showing its maximum at 1 hour (Fig. 26).

B.1. Aptamers binding to ICAM-1 on HNEpC and HEK293 by fluorescence visualization

B.1.1. Preparation of fluorescently labeled RNA aptamers

RNA aptamers modified with a spacer (AAACAAACAAAC; SEQ ID No. 235) and a sense-binding sequence at the 3' end ( GUAUGGCGGUCUCCAACAGG; SEQ ID No. 236 ) were synthesized as previously described in Section A.1.

5'-GGGUGCAUCGUUUACGCGCAACAUAAAAAUUUAAAGUGCUCAGUUGUCAAUCUAUGACUGCUGCUGAGGAAGGAUAUGAG AAACAAACAAAC GUAUGGCGGUCUCCACAGG -3' (SEQ ID No. 229).

A sense-binding sequence was added to anneal to a 6-FAM-labeled fluorescent antisense oligonucleotide. Prior to each binding assay, the NAS-FAM antisense oligonucleotide (5'6-FAM/CCTGTT GGAGACCGCCATAC-3' (SEQ ID No 230)) was mixed with an equimolar concentration of the modified RNA aptamer in 1x selection buffer and incubated at 37°C for 15 minutes.

B.1.2. Preparation of HNepC and Hek293 cells

Prepare HNepC and HEK293 cells according to the procedure outlined in Section A.2, but seed them at a density of approximately 50% onto 12 mm glass coverslips (FisherScientific, catalog number 12-545-82) immersed in culture medium in the wells of a 24-well plate one to two days before assay.

B.1.3. Binding of fluorescently labeled aptamers to cells

Aspirate culture medium from HNepC culture. Then, apply an aliquot of 150 μL of aptamer/NAS-FAM antisense mixture prepared as described in Section B.1 to the cells and incubate at 37°C and 5% _CO2 for 15 min with gentle stirring every 5 min. Aspirate unbound RNA aptamers and wash HNepC three times with 150 μL of 1x selection buffer preheated at 37°C. Remove the coverslip and immerse the cells in a drop of selection buffer on a glass microscope slide. Monitor cell fluorescence using a Nikon reverse fluorescence microscope and a FITC fluorescence filter for up to approximately 1 hour. Image the cells using a Nikon D7500 camera at an exposure of 1/30 sec (see Figures 6A and 6B). Follow the same procedure using HEK293 cells (see Figures 6C and 6D). As shown in Figures 6A and 6B, significant fluorescence was observed when the labeled aptamer was incubated with HNepC, while no fluorescence was detected with HEK293 cells (Figures 6C and 6D), confirming that the aptamer has a stronger binding affinity for surface markers on the surface of HNepC (e.g., ICAM-1) compared to the markers on HEK293 cells.

B.2. Visualization of viral inhibition on H1-HeLa cells using fluorescent viral inhibition assays

RNA aptamers Nas.R-2 and Nas.R-8, which bind to ICAM-1, were tested in a viral inhibition assay to demonstrate their efficacy in inhibiting rhinovirus infection, compared to the negative control aptamer (Figs. 7A to 7H).

B.2.1. Adaptation to body temperature incubation and viral infection

H1-HeLa cells in RPMI+2% fetal bovine serum were seeded at 1 x ^10⁵ cells/mL and 1.0 mL/well into 24-well plates. The inoculation medium was aspirated, and 0.5 mL of each aptamer was added to the host cell wells at 40 μM. The host cells were incubated at 33±2℃ and 5±3% _CO₂ for 30±5 minutes. 0.5 mL of rhinovirus type 14 at ^10³ _TCID⁵⁰ /well was added to the host cell wells without aspiration. The host cells were incubated at 33±2℃ and 5±3% _CO₂ for 120±10 minutes. The host cell wells were aspirated, and the cells were replenished with 1.0 mL of each aptamer in cell culture medium, then returned to 33±2℃ and 5±3% CO₂ for _incubation . After 18±1 hours, the cells were replenished with 1.0 mL of 2X aptamer in the cell culture medium and incubated at 33±2℃ and 5±3% _CO2 for 12±1 hours.

B.2.2. Quantitative Analysis of Viral Inhibition

After the total incubation period, the host cell plates were frozen overnight at -60°C to -90°C and then thawed at ambient temperature. The contents of each well were harvested and centrifuged at 2,000 rpm for 10 minutes. The supernatant from each harvest was collected, serially diluted in cell culture medium, and inoculated onto fresh H1-HeLa cells to determine the amount of infectious virus using a ₅₀ % TCID50 assay. The average yield of virus from control wells treated only with cell culture medium was used to calculate the viral inhibitory activity (Log ₁₀ decrease) for each aptamer.

B.2.3. Table 1: Results

aptamers Log virus potency reduced reduce(%) Nas.R-2 2.08 99.2 Nas.R-8 1.33 95.3

Figures 7A through 7H show the results as images. If the TRITC-labeled virus can infect cells, the cells are marked in red in the fluorescence images. The location of cells in the fluorescence images is marked with arrows based on the corresponding locations in the bright-field images. Infection may not be seen using the Nas.R-2 aptamer (Figure 7A fluorescence image; Figure 7B bright-field image). Infection may be barely visible using the Nas.R-8 aptamer (Figure 7C fluorescence image; Figure 7D bright-field image). Cells infected with the negative control aptamer (Figure 7F bright-field image) appear red (Figure 7E), and Figures 7G and 7H show control cells not infected with the virus (Figure 7G fluorescence image; Figure 7H bright-field image).

C. Binding affinity determined by surface plasmon resonance (SPR)

C.1. Immobilization of RNA aptamers in gold chips

RNA aptamers Nas.R-1, Nas.R-2, Nas.R-4, Nas.R-8, and a negative control were immobilized on the surface of a gold chip. Briefly, the RNA aptamers were dissolved in 1x PBS buffer supplemented with 10 mM EDTA. Then, an aliquot of 20 μL of this solution was added to 3.375 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in a 1.5 mL tube. Next, an aliquot of 13.5 μL of cystamine-imidazole solution was added to the RNA aptamer and EDC solutions, followed by mixing and centrifugation. The supernatant was removed, and an additional 54 μL of 100 μM imidazole (pH 6.0) aliquot was added. The solution was incubated overnight at room temperature. Finally, unincorporated cystamine and imidazole were removed using an RNA removal column.

After conjugating the cystamine moiety with an aminophosphate bond at the 5' phosphate group, the aptamer was immobilized on a gold chip by depositing aliquots of a 10 nL aptamer solution at a concentration of 10 μM onto the chip surface. The gold reduced the cystamine to a pair of thiols, and then catalytically reduced the thiols, resulting in covalent bonds between the gold surface and the thiols of the modified aptamer.

C.2. Surface Plasmon Resonance (SPR) Procedure

A solution of 200 μM ICAM-1 protein or human serum albumin was flowed through a gold chip at a concentration of 250 nM and a flow rate of 50 μL/min using an Openplex surface plasmon resonance system (Horiba, Kyoto, Japan). Thus, the association phase lasted 4 minutes post-injection, followed immediately by the dissociation phase. The total resonance of the negative control aptamer was subtracted from the total resonance observed for each candidate aptamer. The results correspond to the resonance contribution due to protein-aptamer binding. Figure 8 shows the observed binding curves of 250 nM exogenous ICAM-1 with parental aptamers Nas.R-1, Nas.R-2, Nas.R-4, and Nas.R-8. All aptamers showed binding peaks after approximately 250 minutes, with the strongest binding observed for Nas.R-2. Figure 9 shows the results from a comparative experiment using 250 nM human serum albumin, which did not show any significant binding.

The value of kd(koff) is calculated by fitting the curve to equation [1]:

x’～-kd*x   [1]

Where x is the resonance caused by binding, and x' is the derivative of that value at each time point captured on the dissociation curve. The kd value is then used to determine the ka value by using equation [2]:

x’～ka*Rmax*c-(ka*c+kd)*x [2]

Rmax is the maximum resonance caused by the observed binding, and c is the concentration of the injectable agent. Finally, the dissociation equilibrium constant kD was calculated as the ratio of kd to ka (see Table 2). The low nanomolar kD values obtained for different aptamers confirm the strong binding affinity of this molecule for ICAM-1 and verify the aptamer selection process described in Example 1. As used herein, “kd” refers to the dissociation rate, “ka” refers to the association rate, and “kD” refers to the dissociation equilibrium constant.

Table 2. Binding coefficients of Nas.R-1, Nas.R-2, Nas.R-4 and Nas.R-8 on 250 nM exogenous ICAM-1 .

aptamers Nas.R-1 Nas.R-2 Nas.R-4 Nas.R-8 kd, [1/s] 1.27E-02 1.42E-02 2.25E-02 2.63E-03 ka, [1/Ms] 1.97E+05 2.02E+05 5.08E+05 9.27E+04 kD, [M] 6.44E-08 7.02E-08 4.43E-08 2.84E-08

D. Aptamer binding specificity

As described in Example 1, anti-selection was performed using HEK293 cells during the selection process. HEK293 cells do not express the ICAM-1 receptor, but they do express the associated receptor proteins ICAM-3 and ICAM-5. For certain sequences, such as Nas.R-2 (SEQ ID NO:2), the affinity for nasal cells was significantly higher than that for HEK293 cells. Not wishing to be bound by theory, given the presence of ICAM-5 and ICAM-3 on HEK293 cells, it is clear that the selected aptamer binds to an epitope from the ICAM-1 receptor protein region, which is sequence-different from those from the ICAM-5 or ICAM-3 receptors. Figure 10 shows the sequence alignment of the extracellular domains of ICAM-1 (SEQ ID NO: 214), ICAM-3 (SEQ ID NO: 232), and ICAM-5 (SEQ ID NO: 263), highlighting regions that may generate ICAM-1-specific binding. An asterisk (*) indicates a position where amino acid identity is completely conserved, a colon (:) indicates a highly conserved amino acid property with a score greater than 0.5 on the PAM 250 matrix, and a period (.) indicates an amino acid with weak similarity and a PAM 250 score less than 0.5 (Schwartz RM, Dayhoff M. Matrices for detecting distant relationships. In the following reference: Dayhoff M, ed. Atlas of Protein Sequence and Structure. Supplement 3. Vol. 5. National Biomedical Research Foundation; Silver Spring, MD: 1978. Pp. 353–358). The PAM score refers to the probability of mutation occurring, not the similarity of traits; therefore, the definition is a combination of trait conservation and probability of occurrence. For aptamer-protein binding, epitopes are clearly fully conserved when complete amino acid identity exists in the region. However, epitopes may also be conserved when different amino acids are present but traits are retained (positive or negative charge is the most important trait), while epitopes are unlikely to be conserved when there is only weak similarity or no similarity at all.

Rhinovirus binds to the N-terminal Ig-like C2-type 1 domain of the ICAM-1 receptor. Given the selection strategies, including elution with human rhinovirus particles and anti-selection against HEK293 cells, it is clear to those trained in the art that a well-selected library of aptamers will be enriched in aptamer sequences that bind not only to the extracellular domains of the ICAM-1 receptor but also specifically to the N-terminal Ig-like C2-type 1 domain.

Figure 11 shows a fold comparison of aptamer frequencies across the last three selection rounds applied during aptamer selection. Data are expressed as the frequency of a single aptamer sequence selected for nasal cells divided by the frequency of the same sequence observed in the selection for HEK293 cells. For aptamers Nas.R-2, Nas.R-1, and Nas.R-17, no sequences were observed in the selection for HEK293 cells (legend indicates selection round). That is, at least for the subsamples of sequences observed during next-generation sequencing, these sequences were observed at a high frequency in selection round 14 for nasal cells, but were not observed at all in the selection for HEK293 cells.

To avoid being bound by theory, aptamers that did not show frequency enrichment on nasal cells compared to HEK293 cells should be considered as aptamers that are unlikely to inhibit HRV binding. Figure 12 depicts the sequences in selection round 14, all of which showed higher enrichment levels in HEK293 positive selection than in nasal cell positive selection. These aptamers are expected to bind to regions of the ICAM-1 receptor that are not at the N-terminus and have considerable sequence identity with the ICAM-3 or ICAM-5 regions.

B.2.4. Binding of ribonucleotide parental aptamers compared to truncated aptamers

To compare the binding specificity and affinity of ribonucleotide aptamers, binding assays were performed on human nasal epithelial cells (HNEpC) and the rhabdomyosarcoma cell line (RH-30) as a control, using both full-length parental and truncated aptamers. The fluorescein-amidine (FAM)-conjugated RNA aptamers were incubated at 500 nM for 4 h at 37 °C and 5% _CO2 in 24-well plates. At specific time points (0.5 h, 1 h, 2 h, 4 h), cell culture medium was aspirated, and the wells were washed three times with 1X selection buffer. To elute and lyse the cells, 200 μL of 6 M urea was added to the wells, and the plates were incubated at 37 °C for 5 min. The urea-aptamer solution was transferred to a 96-well microplate reader, and absorbance was measured at 495 nm. Samples were measured in triplicate, and the results are shown in Table 3.

B.2.3. Table 3: Binding of truncated aptamers compared to their corresponding full-length parental sequences .

Table 3 summarizes the maximum binding rates to HNEp cells. The truncated aptamers (Nas-R.2.1/SEQ ID NO:237, Nas-R.4.1/SEQ ID NO:239, and Nas-R.8.1/SEQ ID NO:241) all showed significant improvements in binding compared to their full-length parental sequences (SEQ ID NO:2; SEQ ID NO:4 and SEQ ID NO:8). The binding efficacy of the truncated aptamers Nas-R.4.1 (SEQ ID NO:239) and Nas-R.8.1 (SEQ ID NO:241) was more than twice that of the parent aptamers (SEQ ID NO:4; SEQ ID NO:8), and for the truncated aptamer Nas-R.2.1a (SEQ ID NO:237), more than three times the binding efficacy was observed compared to the parent aptamer (SEQ ID NO:2).

Example 3. Sequence similarity analysis .

Alignment was performed on SEQ ID NO:101 to SEQ ID NO:200 using Align X software, a component of Invitrogen's Vector NTI Advanced 11.5.4. Several sequence groups showed at least 90%, at least 70%, or at least 50% nucleotide sequence identity, as illustrated in the example alignments in Figures 13, 14, and 15, which show the results of DNA aptamer analysis. Figure 13 shows the alignment of the central regions of the sequences Nas.D-3 (SEQ ID NO:103) and Nas.D-88 (SEQ ID No:188). The sequences shown (SEQ ID No:247 and SEQ ID No:248) begin at position 18 of the parental sequence, with alignments shown in gray and non-alignments in white. Figure 14 shows the alignment of the central regions of the sequences Nas.D-45 (SEQ ID NO: 145) and Nas.D-8 (SEQ ID NO: 108), and the central regions of the sequences Nas.D-47 (SEQ ID NO: 147) and Nas.D-78 (SEQ ID NO: 178). The sequences shown (SEQ ID NO: 249 and SEQ ID NO: 250, and SEQ ID NO: 251 and SEQ ID NO: 252) begin at position 18 of the parental sequence, where alignments are shown in gray, non-alignments in white, and free positions are indicated by "-". Figure 15 shows a comparison of the central regions of the sequences Nas.D-13 (SEQ ID NO:113) and Nas.D-97 (SEQ ID NO:197), Nas.D-31 (SEQ ID NO:131) and Nas.D-93 (SEQ ID NO:193), Nas.D-39 (SEQ ID NO:139) and Nas.D-82 (SEQ ID NO:182), Nas.D-61 (Seq ID NO:161) and Nas.D-91 (SEQ ID NO:191), as well as the central regions of the sequences Nas.D-87 (Seq ID NO:187) and Nas.D-94 (SEQ ID NO:194). The sequences shown (SEQ ID NO:253 and SEQ ID NO:254, SEQ ID NO:255 and SEQ ID NO:256, SEQ ID NO:257 and SEQ ID NO:258, SEQ ID NO:259 and SEQ ID NO:260, and SEQ ID NO:261 and SEQ ID NO:262) begin at position 18 of the parental sequence, where aligned sequences are shown in gray, unaligned sequences in white, and free positions are indicated by "-". For simplicity, only the central variable region of the aptamer is included in these alignments. Therefore, oligonucleotides having at least 50%, at least 70%, or at least 90% nucleotide sequence identity with sequences showing efficient binding to ICAM-1 are expected to have similar binding efficiency, for example, Nas.D-8 (SEQ ID NO:108).

Example 4. Motif analysis and prediction of secondary structure .

Aptamers bind to target molecules based on the shape they form that has the lowest free energy. This lowest free energy shape varies with homology between regions within a single-stranded sequence. These homologous regions fold over each other, resulting in the secondary and tertiary shapes of the aptamer, which are crucial for binding. We characterize the core properties of these aptamers through a combined analysis of conserved motif sequences and their effects on the predicted structure of the entire aptamer. In this context, a motif is defined as a continuous sequence of nucleotides of a defined length. For this example, we consider each of the six potentially overlapping nucleotide motifs within random regions of each characterized aptamer.

The frequencies of six-nucleotide motifs from random regions of preceding aptamers (Nas.R-1, Nas.R-2, Nas.R-4, and Nas.R-8) in all sequences (nasal epithelial cell division repertoire) from selection round 14 were determined. The average motif frequency was then subtracted from the frequency of each motif, and this value was divided by the standard deviation of all motif frequencies in that selection round to obtain the Z-value for each motif. Clearly, sequences containing high-frequency motifs also bound to the target molecule.

The prediction of aptamer secondary structures was performed using Vienna RNA Websuite (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi.Gruber AR, Lorenz R, Bernhart SH, R, Hofacker IL; Nucleic Acids Research, Volume 36, Issuesuppl_2, July 1, 2008, Pages W70-W74, DOI:10.1093/nar/gkn188) and motifs were highlighted within these structures.

A. Analysis of the role of conserved motifs in the structure of aptamers Nas.R-1/Nas.D-1 :

The results of the motif analysis are shown in Figure 16. This figure provides overlapping six-nucleotide motifs, including random regions of the aptamer, continuously along the x-axis. The y-axis provides statistical significance (Z-value) for each motif in the library. The Z-value is calculated by subtracting the mean frequency of all motifs in the library from the observed frequency of this motif, and then dividing the difference by the standard deviation of all motifs in the library. Therefore, a Z-value of 2 indicates the frequency of this motif in the library that is two standard deviations greater than the mean of all motifs.

The sequences AAACAAAAAGA and UAAAAAUCA are conservatively represented by levels two standard deviations above the mean. Figure 17 shows the predicted structures of the lowest free energy for the Nas.R-1 aptamer and the common sequence.

SEQ ID NO:201：5'-AAACAAAAAGA-3'

SEQ ID NO:202：5'-UAAAAAUCA-3'

It is also anticipated that sequences containing any of these motifs will bind to ICAM-1 and be included as parental embodiments of the invention. The conclusions drawn in this embodiment regarding conserved motifs in RNA sequences will also apply to DNA sequences (Figure 16). Therefore, any sequence containing the corresponding deoxyribonucleotide motif...

SEQ ID NO:203：5'-AAACAAAAAGA-3'

SEQ ID NO:204：5'-TAAAAATCA-3'

It is also included as a parent implementation plan.

B. Analysis of the role of conserved motifs in the structure of aptamers Nas.R-4/Nas.D-4 :

The structural role of conserved motifs within aptamer Nas.R-4 was analyzed in the same manner as described for Nas.R-1, and the conclusions drawn in this example regarding conserved motifs in RNA sequences will also apply to DNA sequences. Figure 19 provides a summary of the motif analysis for aptamer Nas.D-4. Thirteen nucleotide motifs exist at frequencies greater than two standard deviations from the overall average motif frequency in the selected library, namely...

SEQ ID NO 205: 5'-AUAAAAAUUUAAA-3' and the corresponding deoxyribonucleotide motif:

SEQ ID NO 206: 5'-ATAAAAATTTAAA-3'.

It is also anticipated that sequences containing this motif will bind to ICAM-1 and be included as parental embodiments of the present invention.

C. Analysis of the role of conserved motifs in the structure of aptamers Nas.R-8/Nas.D-8 :

The structural role of conserved motifs within aptamer Nas.R-8 was analyzed in the same manner as described for Nas.R-1 and Nas.R-4. The conclusions drawn in this embodiment regarding conserved motifs in RNA sequences will also apply to DNA sequences. Figure 21 provides a summary of the motif analysis for aptamer Nas.D-8. Twelve nucleotide motifs were present at frequencies greater than two standard deviations from the overall average motif frequency in the selected library.

SEQ ID NO:207：5'-GUAAAAAUUAAA-3'

and the corresponding deoxyribonucleotide motif:

SEQ ID NO 208:5'-GTAAAAATTAAA-3'

It is also anticipated that sequences containing this motif will bind to ICAM-1 and be included as parental implementation schemes.

D. Analysis of common motifs in the aptamer library :

A search was conducted on common motifs within the top 100 sequences based on frequency (see Figure 18). The leading motifs identified were SEQ ID NO:209 and SEQ IP NO:210, showing significant deviations from a random distribution.

SEQ ID NO:209：5'-GUAAAAAAA-3'

SEQ ID NO:210：5'-UNAGCANUUU-3'

Oligonucleotides containing the motif SEQ ID NO:209, SEQ ID NO:210, or both are included as parental embodiments of the present invention. Similarly, any sequence containing the corresponding deoxyribonucleotide motif is also contemplated.

SEQ ID NO:211：5'-GTAAAAAAA-3'

SEQ ID NO:212：5'-TNAGCANTTT-3'

It is incorporated in conjunction with ICAM-1 and included as a parent embodiment of the present invention.

E. Preparation of truncated aptamers

E.1.: Identification of suitable truncated sequences

To identify more effective aptamers, oligonucleotides of SEQ ID NO:1 to SEQ ID NO:200 were truncated to the major effective region determined by theoretical analysis. At 37°C, full-length parental aptamers typically flowed in possible shapes. The possible shapes that a single sequence could form were characterized, and the relative proportion of each shape at a given temperature was determined using online software provided by the ViennaRNA Web Services, created by the Theoretical Biology Group of the Institute for Theoretical Chemistry at the University of Vienna. Truncated aptamers exhibiting certain of these shapes at the highest possible stability levels were then identified and synthesized, as disclosed above in Example 2A.1. If desired, single nucleotide substitutions were introduced to stabilize the shape. These truncated, optimized aptamers were then subjected to binding retesting, thus laying the foundation for hypotheses regarding the interaction between aptamer shape and function. The truncated aptamers are designed to retain all the properties of the parent aptamers, particularly exhibiting binding affinity for intercellular adhesion molecule-1 (ICAM-1) and the ability to reduce the binding of one or more human rhinoviruses to ICAM-1. Suitable truncated aptamer sequences comprise about 30 to about 60 nucleotides from the parent aptamer sequence and preferably include at least one conserved motif from the conserved motifs disclosed in SEQ ID NO:201 to SEQ ID NO:212. The resulting truncated sequences are preferably capable of forming the stable secondary structures disclosed herein to serve as aptamers capable of reducing the binding of one or more human rhinoviruses to ICAM-1 over time.

The four aptamer sequences identified in the preceding binding-specific process (Example 2) showing high binding affinity for ICAM-1, Nas.D-2 (SEQ ID NO: 102), Nas.D-4 (SEQ ID NO: 104), Nas.D-5 (SEQ ID NO: 105), and Nas.D-8 (SEQ ID NO: 108), were used as example parental sequences. Deoxyribonucleotide aptamers were chosen for the experiments due to their higher stability compared to ribonucleotide aptamers. The parental sequences were analyzed, and the stable secondary structure based on the lowest free energy was visualized.

Figure 20A illustrates the preferred secondary structure of the parental oligonucleotide of SEQ ID NO:104. The central hairpin structure represents a promising binding candidate for binding to the ICAM-1 receptor. The nucleotides identified in the dashed box are identified to form the truncated aptamer sequence SEQ ID NO:244. Figures 20B and 20C show the truncated oligonucleotide of SEQ ID NO:244 appearing in two different stable secondary structures. Both secondary structures include a common structural concept of a hairpin forming a central loop comprising 15 to 19 unpaired/unhybridized nucleotides (counting from the 5′ end), followed by a double-stranded stem comprising 9 hybridized base pairs in the first secondary structure (Figure 20B) and 9 hybridized base pairs divided into 4 base pairs, a second inner loop containing 5 unpaired/unhybridized nucleotides, and a second double-stranded stem comprising 6 hybridized base pairs in the second secondary structure (Figure 20C). Both secondary structures show unpaired/unhybridized 3′ and 5′ ends. Both variants of the secondary structure are stable and exhibit a common structural concept.

Figure 22A illustrates the preferred secondary structure of the parental aptamer of SEQ ID NO:108. The hairpin structure formed near the 3′ end (marked with a dashed box) represents a promising binding candidate for binding to the ICAM-1 receptor. Therefore, the uninteresting portion of the parental sequence is truncated, resulting in the truncated aptamer of SEQ ID NO:246. The hairpin comprises a central first loop containing 10 unpaired/unhybridized nucleotides, a first double-stranded stem containing 5 hybridized base pairs, a second inner loop containing 3 unpaired/unhybridized nucleotides, a second double-stranded stem containing 3 hybridized base pairs, and unpaired/unhybridized 3′ and 5′ ends (Figure 22A). Figure 22B illustrates a secondary structure that can be achieved through a small mutation in the sequence. By changing T to C at position 15 to form another G-C pair, the central loop is reduced to 8 unpaired/unhybridized nucleotides. The first double-stranded stem still contains 5 hybridized base pairs, while the second loop is reduced to only one unpaired/unhybridized nucleotide at position 10. Therefore, a larger second double-stranded stem, including 5 hybrid base pairs, can be formed by another T-to-C mutation at position 8. The unpaired/unhybridized 3′ and 5′ ends remain unchanged (Figure 22B).

F. Detailed analysis of the secondary structure of truncated aptamers

F.1.: Preparation of mutant aptamers based on truncated aptamer NasD-4.1

The secondary structure motif and its correlation with binding affinity to ICAM-1 were analyzed in detail using the NasD-4.1 truncated aptamer (SEQ ID NO: 244). The importance and/or potential correlation of individual motifs and sequence identity were analyzed using four selection rounds targeting nasal cells, and binding to the ICAM-1 receptor was analyzed using both the original and mutant aptamers (with a mutation load of 9% per nucleotide within the aptamer), as disclosed above according to C.4.3. To facilitate amplification between selection rounds, 9 nucleotides (ATCTAATAA) were added at the 5′ end and 8 nucleotides (AAAAACCC) at the 3′ end to avoid disrupting the predicted secondary structure of the aptamer. Mutation events were limited to the Nas-4.1 aptamer (SEQ ID NO: 244) shown in Figure 20C. The libraries from all four selection rounds, as well as the original unselected library, were analyzed by next-generation sequencing (NGS). A list of all possible single and double mutations within 37 nucleotides was generated, and the frequency of each mutant sequence was extracted through a selection process. The proportion of any given sequence in the library in a selection round was considered a measure of the sequence's efficacy in binding to the ICAM-1 receptor. To minimize the potential impact of PCR error on the interpretation of these results, the average frequency of each sequence in selection rounds 3 and 4 was also calculated and divided by the corresponding frequency of that sequence in the original library (selection round 0). One was subtracted from the divisor to reduce the fraction, thus directly indicating the proportion change (“proportion”) of each sequence.

F.2.: Correlation between aptamer efficacy and structural motifs

Analysis of the effects of sequences on aptamer efficacy may be limited, but the reason different sequences behave this way is that they have different effects on aptamer structure. These differences manifest in the presence or absence of charged groups that interact with the ICAM-1 receptor and the differences in distance between these interactions. The analysis of all sequences constituting a given mutant load can be viewed as an examination of the sequence space at the level surrounding the non-mutant sequence. Therefore, aptamer sequences were transferred to their structural motifs based on the code published in P. Danaee et al. (Nucleic Acids Research, Vol. 46, No. 11, June 20, 2018, pp. 5381–5394) and based on the predicted aptamer structures disclosed herein. Transferring the 4.1 aptamer sequences to the structural code allows for comparison of structural effects independent of the aptamer nucleotide sequence.

The structural code for aptamer 4.1 is

5'-EEEEEEEEEESISSSSSSISSSSHHHHHSSSSIIIISSSSSSISEEEEEEEEEE-3'

The database of such structural information strings was then evaluated to identify the motifs that showed the strongest statistical significance for favorable selection. The MEME software suite (TLBailey et al.; Nucleic AcidsResearch , 43(W1):W39-W49, 2015) was used to explore shared motifs and their relationship to structural codes, as shown above. Regarding efficacy (proportionality) sequences, the top 500 double-mutant sequences were compared with the bottom 500. Thus, the most consistent structural motifs present in the top 500 and bottom 500 sequences were identified. The analysis stopped after identifying three motifs. Figure 27A shows the results. The relative height of the structural code is a measure of information certainty, i.e., it shows the degree of certainty in observing a particular code at a specific location. The leading motif was IISS ^S / _E EEEEE. This implicitly refers to the dangling end of the aptamer's 3' end, taking into account the stem structure near the 5' side of E. Other motifs of interest are the hairpin bend at the base of the aptamer, followed by a stem structure on the 3' side, and a dangling 5' end of 12 nucleotides. The distribution of these motifs in the aptamer is provided in Figure 27B, which shows examples of some sequences included in the statistically significant categories identified above. The presence of the second motif (large gray square) indicates the need for a hairpin/stem structure, but the position of these structures in the aptamer is not entirely fixed. The first motif (black) is observed only at the 3' end, while the third motif (small gray square) is observed at both the 5' and 3' ends. The third motif does not imply anchorage, as it simply represents a continuous string of E (dangling ends). When this motif ends before the apse, it still implies that the aptamer has a dangling end extending to the terminal nucleotide. The same conclusion applies to the first motif (black). If this motif is present, all positions between it and the 3' end will also be dangling ends.

In summary, structural analysis identified three important regions of interest within the aptamer.

1.) 3' Suspended end, including the connection between the suspended end and the upstream stem structure.

2.) The hairpin structure, whose position within the aptamer is not completely fixed.

3.) 5' suspended end.

F.3.: Correlation between aptamer efficacy and structural motifs

As shown above, effective aptamers include both ends as overhangs and an inner stem/hairpin structure. Sequences associated with the identified motifs were analyzed, and accumulations of interest were found in the structural motifs at the 3′-overhang. Figure 28 shows a portion of a graph visualizing the frequency (average proportion) of aptamers including said sequences. All aptamers found to be highly present (meaning highly effective) included the motif GAGGYYZ in their 3′-overhang sequences. The average proportion observed for aptamers with the sequence GAGGYYZ was 0.23, while the average proportion observed for aptamers not displaying a motif at that position was -0.17.

F.4.: Binding analysis of the mutant Nas-4.1 sequence

The aptamers disclosed herein were selected for SELEX selection at 37°C. Furthermore, all experiments relating to aptamer performance performed on cells grown in culture were also conducted at 37°C, as this is the optimal temperature for cell growth and corresponds to body temperature. However, the aptamers are intended to act on the inner surface of the nasal cavity in vivo, and the nasal cells exhibit a slightly lower average temperature of 33°C. Thus, it is important to ensure that the aptamers of the present invention function at both temperatures. Therefore, binding analysis was performed at 37°C and 33°C using an isothermal titration calorimetry (ITC) device, following the user instructions for the NasD-4.1 aptamer (SEQ ID NO: 244). Specific mutations in the sequences (SEQ ID NO: 264-270) were tested to analyze the effects of temperature and secondary structure. NasD-4.1 aptamer at a concentration of 600 nM was injected 4.88 μL into 193 μL of 100 nM ICAM-1 receptor protein in 11 fractions. The same concentration of mutant aptamer was injected into the buffer, and the buffer was injected into the same amount of protein as a control. ITC experiments were performed at 33 °C and 37 °C in potassium phosphate buffer.

The following aptamers were tested, with mutations shown in bold:

NasD-4.1：5′-GTG CTC AGT TGT CAA TCT ATG ACT GCT GCT GAG GAAG-3 (SEQ IDNO.244)

State 1:

State 2:

State 3:

State 4:

State 5:

Status 6:

Status 7:

The dissociation constant (kD) was measured against the original/unmutated Nas-D4.1 aptamer, a direct measure of the binding affinity of the mutant aptamer. kD values are expressed in nM. Higher values than those for the Nas-D 4.1 aptamer indicate lower binding affinity of the mutant, and vice versa. The results are shown in Table 4.

Table 4

The mutation in the state 1 aptamer (SEQ ID NO. 264) is located near the 5′ end. Structural prediction revealed that this structure is stable at both temperatures, and the GAAGYYZ motif is located at the dangling 3′ end. Predicted secondary structures of all mutant aptamers were generated at 33 °C and 37 °C using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). Figure 29 shows the most likely secondary structure of mutant state 1, where grayscale indicates the probability of the nucleotide being in the indicated structure, with darker shades indicating higher probability. No 5′ dangling end was observed compared to the unmutated NasD-4.1 aptamer (SEQ ID NO. 244). This may explain the lower binding affinity of the mutant under in vivo conditions at 33 °C. The mutation in the aptamer of state 2 (SEQ ID NO. 265) is also located near the 5′ end, but compared to the control NasD-4.1 aptamer (SEQ ID NO. 244), it aimed to correct the secondary structure of the aptamer by fixing the GAGGYYZ motif in half of the stem region. However, the most probable predicted structure (Fig. 30) shows that the mutation did not change the secondary structure, and the GAGGYYZ motif remained at the suspended 3′ end, which is very consistent with the almost unchanged binding affinity under in vivo conditions. To understand the reduced binding affinity of mutant aptamer state 2 at 37°C, the preferred secondary and tertiary structures formed under equilibrium conditions were again determined using online software provided by ViennaRNA WebServices. Fig. 31 shows the results, where hybridized nucleotides are shown in dark gray, while unhybridized nucleotides are shown in light gray. The most probable structure 1 (Fig. 31A) is the structure also calculated in Fig. 30. The second most probable structure 2 (Fig. 31B) differs in that a larger inner loop is formed, and the second stem includes a fifth base pair. Therefore, the dangling end is shortened, and the GAGGYYZ motif portion is located in the stem region. In the third possible secondary structure (Fig. 31C), a second inner loop and a third stem are formed, resulting in only one nucleotide dangling at the 5′ end, but the complete GAGGYYZ motif dangling at the 3′ end. Fig. 32 shows the distribution of the three secondary structure variants shown in Fig. 31 at 33°C (Fig. 32A) and 37°C (Fig. 32B). At both temperatures, structure 1 (shown in Fig. 31A) is dominant. However, at 37°C, the share of structure 3 (shown in Fig. 31C) is significantly larger than at 33°C. Therefore, at 37°C, the share of structure 2 (shown in Fig. 31B) is significantly smaller than at 33°C. This difference in the share distribution of different secondary structure variants leads to a loss of binding affinity for mutant aptamer state 2. Unwilling to be bound by theory, it is believed that the missing dangling 5′ end has a negative impact on the binding affinity of the entire aptamer, similar to what has been observed in mutant aptamer state 1.

The mutation in the state 3 aptamer (SEQ ID NO. 266) is at the first position of the GAGGYYZ motif. A decrease in binding affinity was observed at both temperatures. It appears that the mutation has a smaller effect on aptamer performance at in vivo temperatures. The mutation in the state 4 mutant aptamer (SEQ ID NO. 267) is at the fourth position of the GAGGYYZ motif. Surprisingly, the binding affinity of the state 4 mutant aptamer was almost unaffected at 37 °C, while a decrease in binding affinity was observed at 33 °C. Secondary structure prediction revealed a four-base hybridization in the mutant GAGTAAG motif (as expected by the mutation) and the formation of a stem that results in lower binding affinity under in vivo conditions (Fig. 33A). However, at higher temperatures (37°C), mutations in the motif sequence transform the secondary structure into a shape where the GAGTAAG motif is completely unhybridized and suspended at the 3′ end (Fig. 31B), thus becoming more similar to the NasD-4.1 structure. Full functionality of this mutant state was observed in ITC binding experiments at higher temperatures. The grayscale in Fig. 33 indicates the probability of the nucleotide being in the illustrated structure, with darker shades indicating a higher probability. Without being bound by theory, it is believed that the degree of freedom of the 3′-suspended sequence compensates for the potential negative impact of the sequence motif mutation, resulting in a slightly higher binding affinity.

The mutation in the state 5 mutant aptamer (SEQ ID NO. 268) is located in the stem region just before the GAGGYYZ motif. Compared with the control NasD-4.1 aptamer (SEQ ID NO. 244), these mutations did not predict general changes in the secondary structure of the mutant aptamer, and a slight improvement in binding affinity was observed.

In the mutant aptamer of state 6 (SEQ ID NO: 269) and state 7 (SEQ ID NO: 270), the hairpin motif is mutated. In state 6, the hairpin loop is enlarged, which decreases the binding affinity at 37 °C and increases it at 33 °C. Without being bound by theory, it is believed that the size of the hairpin structure is less relevant at 33 °C compared to 37 °C. In mutant aptamer state 7, the hairpin loop is formed from different nucleotides compared to the original aptamer (SEQ ID NO: 244), but the effect on binding affinity at both temperatures is noteworthy.

Combined with experimental results, it was shown that the aptamers of the present invention also possess similar functionality at 33°C, and thus function appropriately for nasal cells in vivo. Performance analysis of the mutant states, particularly at 33°C, also revealed the effect of certain sequence- and/or structural features claimed herein on in vivo performance. For example, nucleotide mutations that do not affect secondary structure did not significantly alter binding affinity (state 5). Similar results were observed with exchange in hairpin loop sequences (state 7). Increasing the size of the hairpin loop (state 6) was considered advantageous under in vivo conditions, as an increase in binding affinity was observed. A significant decrease in binding affinity was observed with mutations in the GAGGYYZ motif (state 3) or with the motif fixed in half of the stem region (state 4). Not wishing to be bound by theory, it was concluded that the aptamers with a completely unhybridized GAGGYYZ motif (injected into the suspended 3′-end region) have higher binding affinity than aptamers with a GAGGYYZ motif that is still effective but includes partial hybridization in the stem region.

Table 5. List of parental ribonucleotide sequences selected for the experiment. All pyrimidine nucleotides were fluorinated at the 2' position of the pentose group.

Table 6. List of parental aptamers of deoxyribonucleotides based on the preceding sequences from the selection experiment .

Table 7. List of conserved motifs .

Table 8. List of truncated aptamers and parental aptamers of ribonucleotides based on the preceding information .

Table 9. List of protein sequences

combination

A. An aptamer composition comprising at least one oligonucleotide consisting of: deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof, wherein the aptamer composition has binding affinity for intercellular adhesion molecule-1 (ICAM-1), and wherein the aptamer is configured to reduce the binding of one or more human rhinoviruses to ICAM-1, and wherein the aptamer composition comprises at least one oligonucleotide forming a secondary structure from a 5′-end to a 3′-end, the secondary structure comprising at least a 5′-overhanging end, a stem, a hairpin loop, and a 3′-overhanging end, wherein the stem is formed between the hairpin loop and the overhanging ends; and

The secondary structure includes at least the structural sequence 5′-EEEYYSSSSYSSSSYHHHHYYYYYYYYSYYYYYEEEE-3′ structure I, where H represents a nucleotide located in the hairpin loop, S represents a nucleotide located in the stem, E represents a nucleotide located in the dangling end, and Y represents the absence of the nucleotide or H, S, E or I, where I represents the inner loop.

B. The aptamer composition according to paragraph A, wherein the hairpin ring comprises at least 5 nucleotides, preferably wherein the hairpin ring comprises at least one purine base, at least one pyrimidine base, or a combination thereof, more preferably wherein the hairpin ring comprises at least three pyrimidine bases, or a combination thereof.

C. The aptamer composition according to paragraph A or B, wherein the last nucleotide in the stem adjacent to the hairpin loop is a pyrimidine base, and wherein the first nucleotide in the stem following the hairpin loop is a purine base or a combination thereof.

D. The aptamer composition according to any one of paragraphs A to C, wherein the last nucleotide in the stem adjacent to the 3′-overhang is a purine base, and wherein the first nucleotide in the stem adjacent to the 5′-overhang is a pyrimidine base or a combination thereof.

E. The aptamer composition according to any one of paragraphs A to D, wherein the last four nucleotides of the 3′-hanging end are purine bases, preferably the last five, six or seven nucleotides of the 3′-hanging end are purine bases.

F. The aptamer composition according to any one of paragraphs A to E, wherein the 5′-suspended end comprises at least 4 nucleotides, preferably 4 to 15 nucleotides, and more preferably wherein the 5′-suspended end comprises at least two purine bases.

G. An aptamer composition according to any one of paragraphs A to F, the aptamer composition comprising an inner ring, a first stem and a second stem, wherein the inner ring is located between the first stem and the second stem, and wherein the inner ring comprises at least one nucleotide, preferably comprising one to ten nucleotides, more preferably one to five nucleotides.

H. The aptamer composition according to any one of paragraphs A to G, wherein the second stem is located adjacent to the suspended end.

I. The aptamer composition according to any one of paragraphs A to H, wherein the stem adjacent to the suspended end comprises at least 3 base pairs, preferably at least 4 base pairs, more preferably at least 5 base pairs.

J. The aptamer composition according to any one of paragraphs A to I, wherein the stem adjacent to the 3′-overhanging end comprises a motif of guanine-cytosine-thymine (GCT) and/or wherein the stem adjacent to the 5′-overhanging end comprises a motif of thymine-cytosine-adenine (TCA).

K. An aptamer composition according to any one of paragraphs A to J, wherein the at least one oligonucleotide comprises a length of 30 to 60 nucleotides, preferably 30 to 55 nucleotides, more preferably 30 to 50 nucleotides, more preferably 30 to 45 nucleotides, more preferably 35 to 40 nucleotides, said length being more preferably truncated from the parent oligonucleotides selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200, more preferably truncated from the parent oligonucleotides selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:10 or SEQ ID NO:101 to SEQ ID NO:110, more preferably truncated from the parent oligonucleotides selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:8 or SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:108.

L. An aptamer composition according to any one of paragraphs A to K, wherein the at least one oligonucleotide comprises a natural or non-natural nucleobase, preferably wherein the non-natural nucleobase is selected from the group consisting of: hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thion-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosine-1-yl, 2-aminoadenine-9-yl, 7-deazo-7-iodoadenine-9-yl, 7-deazo-7-propynyl-2-aminoadenine-9-yl, phenoxazinyl, phenoxazinyl-G-clam, and mixtures thereof.

M. An aptamer composition according to any one of paragraphs A to L, wherein the ribonucleotide derivative or the deoxyribonucleotide derivative is selected from the group consisting of locked oligonucleotides, peptide oligonucleotides, diol oligonucleotides, threonucleotides, hexitol oligonucleotides, azotol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabinose oligonucleotides, 2'-fluoroarabinose oligonucleotides, cyclohexene oligonucleotides, diaminophosphate morpholino oligonucleotides, and mixtures thereof.

N. The aptamer composition according to any one of paragraphs A to M, wherein the nucleotides at the 5' and 3' ends of the at least one truncated oligonucleotide are reversed.

O. An aptamer composition according to any one of paragraphs A to N, wherein at least one nucleotide of the at least one truncated oligonucleotide is fluorinated at the 2' position of the pentose group, preferably wherein the pyrimidine nucleotide of the at least one truncated oligonucleotide is fluorinated at the 2' position of the pentose group.

P. An aptamer composition according to any one of paragraphs A to O, wherein the aptamer has binding affinity for the Ig-like C2 type 1 domain (SEQ ID NO: 215) of intercellular adhesion molecule 1 (ICAM-1), any post-translational modification of the domain, and mixtures thereof.

Q. A personal healthcare composition comprising at least one aptamer composition according to any one of paragraphs A to P.

R. An aptamer composition according to any one of paragraphs A to P or a personal healthcare composition according to paragraph Q, the aptamer composition or the personal healthcare composition being used to inhibit human rhinovirus infection by inhibiting the binding of the human rhinovirus to the intercellular adhesion molecule 1 (ICAM-1) and thereby inhibiting entry into cells within the nasal cavity and pharynx, and/or for the prevention and treatment of symptoms associated with respiratory viral infection, preferably by delivering the composition to the upper respiratory tract.

The dimensions and values disclosed herein should not be construed as strictly limited to the precise numerical values cited. Rather, unless otherwise specified, each such dimension is intended to represent the stated value and the range around which it is functionally equivalent. For example, a dimension disclosed as “40 mm” is intended to represent “approximately 40 mm”.

The values disclosed herein as range endpoints should not be construed as strictly limited to the exact numerical values referenced. Rather, unless otherwise specified, each range of values is intended to represent the referenced values and any real numbers within the range, including integers. For example, a range disclosed as “1 to 10” is intended to represent “1, 2, 3, 4, 5, 6, 7, 8, 9, and 10”, and a range disclosed as “1 to 2” is intended to represent “1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2”.

Unless expressly excluded or otherwise limited, every reference cited herein, including any cross-references or related patents or patent applications, and any patent application or patent claiming priority to or benefiting from it, is incorporated herein by reference in its entirety. Reference to any reference is not an endorsement of it as prior art to any disclosed or protected art herein, nor is it an endorsement of any such invention, either on its own or in combination with any one or more references. Furthermore, where any meaning or definition of a term in this invention conflicts with any meaning or definition of the same term in referenced documents, the meaning or definition given to that term in this invention shall prevail.

While specific embodiments of the invention have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be covered by the appended claims.

Claims (16)

1. An aptamer composition comprising at least one oligonucleotide consisting of: deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof, wherein the aptamer composition has binding affinity for intercellular adhesion molecule-1 (ICAM-1), and wherein the aptamer is configured to reduce the binding of one or more human rhinoviruses to said intercellular adhesion molecule-1 (ICAM-1), and The aptamer composition comprises at least one oligonucleotide forming a secondary structure from a 5′-end to a 3′-end, the secondary structure including at least a 5′-overhanging end, a stem, a hairpin loop, and a 3′-overhanging end, wherein the stem is formed between the hairpin loop and the overhanging end; and The secondary structure includes at least the structural sequence 5′-EEEYYSSSSYSSSSYHHHHYYYYYYYYSYYYYYEEEE-3′ (structure I), where H represents a nucleotide located in the hairpin loop, S represents a nucleotide located in the stem, E represents a nucleotide located in the dangling end, and Y represents the absence of the nucleotide or H, S, E, or I, where I represents the inner loop. 2. The aptamer composition according to claim 1, wherein the hairpin loop comprises at least 5 nucleotides, preferably wherein the hairpin loop comprises at least one purine base, at least one pyrimidine base, or a combination thereof, more preferably wherein the hairpin loop comprises at least three pyrimidine bases, or a combination thereof. 3. The aptamer composition according to claim 1 or 2, wherein the last nucleotide in the stem adjacent to the hairpin loop is a pyrimidine base, and wherein the first nucleotide in the stem following the hairpin loop is a purine base or a combination thereof. 4. The aptamer composition according to any one of the preceding claims, wherein the last nucleotide in the stem adjacent to the 3′-overhang is a purine base, and wherein the first nucleotide in the stem adjacent to the 5′-overhang is a pyrimidine base or a combination thereof. 5. The aptamer composition according to any one of the preceding claims, wherein the last four nucleotides of the 3′-hanging end are purine bases, preferably the last five, six or seven nucleotides of the 3′-hanging end are purine bases. 6. The aptamer composition according to any one of the preceding claims, wherein the 5′-suspended end comprises at least 4 nucleotides, preferably 4 to 15 nucleotides, and more preferably wherein the 5′-suspended end comprises at least two purine bases. 7. The aptamer composition according to any one of the preceding claims, the aptamer composition comprising an inner loop, a first stem and a second stem, wherein the inner loop is located between the first stem and the second stem, and wherein the inner loop comprises at least one nucleotide, preferably wherein the inner loop comprises one to ten nucleotides, more preferably one to five nucleotides. 8. The aptamer composition according to the preceding claim, wherein the second stem is located adjacent to the suspended end. 9. The aptamer composition according to any one of the preceding claims, wherein the stem adjacent to the suspended end comprises at least 3 base pairs, preferably at least 4 base pairs, more preferably at least 5 base pairs. 10. The aptamer composition according to any one of the preceding claims, wherein the stem adjacent to the 3′-overhanging end comprises a motif of guanine-cytosine-thymine (GCT) and/or wherein the stem adjacent to the 5′-overhanging end comprises a motif of thymine-cytosine-adenine (TCA). 11. The aptamer composition according to any one of the preceding claims, wherein the at least one oligonucleotide comprises a length of 30 to 60 nucleotides, preferably 30 to 55 nucleotides, more preferably 30 to 50 nucleotides, more preferably 30 to 45 nucleotides, more preferably 35 to 40 nucleotides, the length being more preferably truncated from the parent oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:200, more preferably truncated from the parent oligonucleotide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:10 or SEQ ID NO:101 to SEQ ID NO:110, more preferably truncated from the parent oligonucleotide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:8 or SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105 and SEQ ID NO:108. 12. The aptamer composition according to any one of the preceding claims, wherein the at least one oligonucleotide comprises a natural and a non-natural nucleobase, preferably wherein the non-natural nucleobase is selected from the group consisting of: hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thion-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosine-1-yl, 2-aminoadenine-9-yl, 7-deazo-7-iodoadenine-9-yl, 7-deazo-7-propynyl-2-aminoadenine-9-yl, phenoxazinyl, phenoxazinyl-G-clam, and mixtures thereof. 13. The aptamer composition according to any one of the preceding claims, wherein the ribonucleotide derivative or the deoxyribonucleotide derivative is selected from the group consisting of: Locked oligonucleotides, peptide oligonucleotides, diol oligonucleotides, threoylose oligonucleotides, hexitol oligonucleotides, azotitol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabinose oligonucleotides, 2'-fluoroarabinose oligonucleotides, cyclohexene oligonucleotides, diaminophosphate morpholino oligonucleotides, and mixtures thereof. 14. The aptamer composition according to any one of the preceding claims, wherein at least one nucleotide of the at least one oligonucleotide is fluorinated at the 2' position of the pentose group, preferably wherein the pyrimidine nucleotide of the at least one oligonucleotide is fluorinated at the 2' position of the pentose group. 15. A personal healthcare composition comprising at least one aptamer composition according to any one of the preceding claims. 16. The aptamer composition according to any one of claims 1 to 14 or the personal healthcare composition according to claim 15, wherein the aptamer composition or the personal healthcare composition is used to inhibit human rhinovirus infection by inhibiting the binding of human rhinovirus to the intercellular adhesion molecule 1 (ICAM-1) and thereby inhibiting entry into cells within the nasal cavity and pharynx, and/or is used preferably by delivering the composition to the upper respiratory tract to prevent and treat symptoms associated with respiratory viral infection.