WO2022208171A1

WO2022208171A1 - Methods for analyte detection

Info

Publication number: WO2022208171A1
Application number: PCT/IB2022/000179
Authority: WO
Inventors: Sam RODRIQUES; Patrick ARNOTT
Original assignee: UCL Business Ltd.
Priority date: 2021-03-31
Filing date: 2022-03-29
Publication date: 2022-10-06

Abstract

This disclosure provides methods and systems detecting and quantifying proteins in a sample using single-stranded nucleic acid aptamers.

Description

METHODS FOR ANALYTE DETECTION

TECHNICAL FIELD

This disclosure relates to methods and systems for proteomic analysis.

BACKGROUND

Many conventional methods for detecting and analyzing analytes, especially proteins, in a biological sample rely, at least in part, on antibodies. Proteomic analysis generally involves immunoassays. However, immunoassay methods possess inherent difficulties, complications, and costs, especially when used in a multiplex format.

More recent technologies that are not antibody-based have begun to emerge. However, they too have drawbacks. For example, some methods employ surface plasmon resonance for detection, which requires highly skilled operators using delicate and complex equipment. Further, current non-antibody based methods have shown high limits of detection (LoD) and functional limits of quantification (FLoQ). This can severely limit their practical use for many purposes.

In one example, free circulating p-Tau proteins have been found to provide a useful measure to diagnose and assess the progression of Alzheimer’s disease, especially in cerebrospinal fluid. However, even in patients with advanced Alzheimer’s, the concentration of free circulating p-Tau in blood is at attomolar levels, which is too low for conventional methods of quantification and detection.

Thus, there exists a need in the art for improved methods for detecting, identifying and quantifying analytes in biological samples.

SUMMARY

The present disclosure provides systems and methods for detecting and quantifying target analytes in a sample using nucleic acid aptamers. These methods and systems do not require the use or development of antibodies for their implementation. Rather, they leverage robust, sensitive, and readily-deployable nucleic acid amplification and/or sequencing techniques for detection. According to methods of the invention, an aptamer that has complementarity with one or more analyte in a sample is bound to a nucleic acid such that when the analyte binds to the aptamer, the nucleic acid is displaced. By amplifying, sequencing and/or detecting the nucleic acid, the presence and/or amount of the analyte in the sample is determined.

Methods and systems of the invention have been shown to provide a limit of detection (LoD) for proteins in a biological sample at a concentration lower than about 40aM and a functional limit of quantification (FLoQ) at a concentration lower than about 2fM. These attomolar and femtomolar concentrations are orders of magnitude lower than conventional non antibody-based protein detection assays. Thus, the presently- disclosed systems and methods are useful in diagnostic assays, especially those in which detection of minute quantities of analytes, such as proteins, is necessary for timely diagnosis and intervention. Moreover, because the LoD and FLoQ are low, the presently-disclosed methods are effective with smaller, more dilute samples, such as saliva, thus leading to improved non-invasive diagnostic tests.

Methods of the invention combine analyte detection with DNA amplification and detection techniques. In a preferred embodiment, the analyte is a protein, but it will be apparent to the skilled artisan that any analyte that functions in accordance with the principles of the invention is subject to detection using methods taught herein. For purposes of exemplification, the following summary will refer to protein detection, understanding that any analyte that binds an aptamer construct as described herein is detectable using methods of the invention.

In short, methods of the invention use target-specific, single-stranded nucleic acid aptamers that are hybridized to a second nucleic acid. The second nucleic acid has regions, both complementary and non-complementary, to the sequence of the aptamer. Thus, when an aptamer encounters a target analyte (e.g., a protein) in a sample, favorable binding kinetics cause the aptamer to interact with the protein, which displaces the second nucleic acid. The released second nucleic acid is then available for amplification (e.g., qPCR), which is used to detect and/or quantify the target analyte in the sample.

A preferred use of the present invention is the detection of proteins or peptides in biological samples, especially samples comprising a heterogenous mix of target and non-target molecules. An exemplary method of the disclosure includes contacting a sample from a subject with a target-specific, single-stranded nucleic acid aptamer that is hybridized to a second nucleic acid with regions complementary to the aptamer. Target proteins in the sample are captured by the aptamers. This capture displaces the second nucleic acid from the aptamer. The second nucleic acid, now free from the aptamer, is amplified for the presence and/or identity of the captured target protein in the sample.

Methods of the invention allow detection of a target protein in a sample at or below a concentration of about 40aM. As will be apparent to the skilled artisan, inventive methods are amenable to quantifying the target analyte. For example, the amount of a target protein can be quantified at or below a concentration of about 2fM.

Methods of the invention can be multiplexed. For example, a sample is contacted with a plurality of different aptamers that are each bound to a different second nucleic acid. Thus, in certain methods, each different target-specific aptamer captures a different target protein and the amplifying step identifies the presence of the different target proteins captured. The amount of these different target proteins can be quantified during the amplification step.

The aptamer can be conjugated to a solid surface and can be attached by any appropriate linker, such as biotin/streptavidin, dioxigenein/anti-dioxigenin, FITC/Anti-FITC, thiol/maleimide, amine/NHS, carbohydrate/lectin and other binding combinations. An aptamer for use in the invention can be any aptamer that binds a DNA or RNA sequence of any length. The aptamer may contain modified nucleic acids, such as peptide nucleic acids, locked nucleic acids, and others. The aptamer may be designed to bind an antibody or fragment thereof. In one alternative, the aptamer is replaced by an antibody and the release strand is displaced by antigen binding to the antibody.

In methods of the invention, nucleic acid barcodes, such as unique molecular identifiers (UMIs) to enable detection of released nucleic acids. This optional feature of the invention enables multiplexing and the ability to trace target analytes to samples and patients from whom they were obtained.

The invention is also applicable to the identification of aptamers useful for binding a known analyte in a sample. The identification is via the released nucleic acid (second) strand as described above and allows the identification of target-specific aptamers from aptamer libraries for use in assays to detect the analyte. For example, an aptamer library is screened according to the invention in order to identify aptamers that optimally detect specific analytes.

Methods of the invention are particularly successful in the detection and/or quantification of a target analyte that is associate with a pathology. The pathology may, for example, be a viral infection and the target protein a viral associated protein. In certain aspects, the viral infection is a respiratory virus, such as a severe acute respiratory syndrome (SARS) virus, such as a SARS coronavirus (e.g., SARS-CoV-2). For example, the invention is useful to detect the spike protein present in the SARS-CoV-2 virus. In certain methods, the sample is contacted with a number of different target-specific, single-stranded nucleic acid aptamer that are each hybridized to a different second nucleic acid and capture a different SARS-CoV-2 spike protein isoform or variant.

The present disclosure also includes methods in which the pathology is, for example, Alzheimer’s Disease (AD) and the target protein is a p-Tau protein. In certain aspects, the amplifying step quantifies the amount of the p-Tau protein in the sample and the method further includes predicting the risk of AD in the subject using the quantified amount of the p-Tau protein.

In certain other aspects, the sample is obtained from one or more of blood, cerebrospinal fluid, semen, fecal matter, saliva, urine, bile, bone marrow, tissue, mucus, tears, and sweat.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a schematic overview of a method for detecting and quantifying proteins in a sample.

FIG. 2 provides a schematic overview of a method for detecting SARS-CoV-2 spike protein in a sample.

FIG. 3 A illustrates the receptor binding domain of the SARS-CoV-2 spike protein interacting with an aptamer of the invention.

FIG. 3B provides a closeup view of the SARS-CoV-2 spike protein receptor binding region interacting with the aptamer.

FIG. 4 provides gels showing the results of an assay to detect the SARS-CoV-2 spike protein in a sample using the aptamer and release strand of the present invention.

FIG. 5 provides gel showing the result of an assay to detect the SARS-CoV-2 spike protein in a sample using the aptamer release strand of the present invention down to 40 aM.

FIG. 6 provides a schematic overview of a method to show that the second nucleic acids are displaced from the aptamer by the SARS-CoV-2 spike protein. FIG. 7 provides emission spectrums showing the disassociation of the second nucleic acids from the aptamers due to the aptamer interacting with the SARS-CoV-2 spike protein.

DETAILED DESCRIPTION

The present invention provides systems and methods for detecting and quantifying analytes in a sample using nucleic acid aptamers. The present systems and methods use target- specific, single-stranded nucleic acid aptamers. Each aptamer is hybridized to a second nucleic acid. The second nucleic acids contain regions both complementary and non-complementary to the aptamers. Thus, when an aptamer encounters, for example, a target protein in a sample, favorable binding kinetics cause the aptamer to interact with the protein, which displaces the second nucleic acid. This released second nucleic acid is then detected. The detection can be via amplification, such as qPCR, followed by sequencing, hybrid capture or any other method for identifying the second nucleic acid. In some embodiments, the liberated second nucleic acid is directly sequenced (i.e., without amplification).

Methods of the invention have been shown to provide a limit of detection (LoD) for proteins in a sample at a concentration lower than about 40aM and a functional limit of quantification (FLoQ) at a concentration lower than about 2fM. These attomolar and femtomolar protein concentrations are orders of magnitude lower than conventional non-antibody -based protein detection assays. The following detailed description and examples are provided to exemplify the invention. The skilled artisan understands that the exemplification provided herein is useful for detection of a variety of analytes via a variety of detection methods.

Fig. 1 provides a workflow for an exemplary method 101 of the disclosure for detecting and/or quantifying one or more target analytes in a sample. A sample is exposed to target- specific aptamers. Each aptamer includes a target-specific single-stranded nucleic acid 106. The aptamer is hybridized to a second nucleic 116 acid at regions complementary to the aptamer 114. The aptamer may be attached to a moiety 110, which can be conjugated to a solid surface 112 to anchor the aptamer. In certain aspects, the aptamer, nucleic acid, and second nucleic acid have non-complementary regions and/or regions where hybridization is prevented, for example, by using modified nucleotide analogues and/or nonstandard nucleobases. The aptamer captures a target analyte 120 in the sample, displacing the second nucleic acid from the aptamer/nucleic acid. The protein-aptamer complex is optionally removed in a wash step 107.

The released, second nucleic acids are then amplified 109 to detect and/or quantify the target protein in the sample. It is only upon displacement from the aptamer that the second, partially complementary nucleic acids become available for an amplification reaction. Thus, amplification of a released second nucleic acid indicates that a target protein was contained in a sample as it bound to the aptamer and displaced the second nucleic acid. Conversely, any aptamer that failed to have its second nucleic acid displaced (i.e., because its target analyte was not in the sample), remains bound to its second nucleic, which is, therefore, not amplified.

In certain aspects, a sample is contacted with a plurality of the same aptamer. Each aptamer captures the same type of target protein. When aptamers associate with a cognate target protein, the second nucleic acids are liberated and subject to detection. In certain aspects, the amplification products for each second nucleic acid can be distinguished (e.g., through the use of unique molecular indices). The number of distinct amplification products are used to quantify the amount of the target protein in a sample. Alternatively or additionally, a number of different aptamers are used, each capturing a different target protein, and each with a different second nucleic acid. In such cases, the amplification products of the different second nucleic acids are used to identify and quantify different target proteins in a sample.

In certain aspects, the second nucleic acids are amplified using a polymerase chain reaction (PCR)-based assay to detect the presence of, and quantify the amount of, second nucleic acids displaced from their aptamers. Examples of PCR-based assays of interest include, but are not limited to, quantitative PCR (qPCR), quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), digital droplet PCR (ddPCR) single cell PCR, PCR-RFLP/real time- PCR-RFLP, hot start PCR, nested PCR, in situ polony PCR, in situ rolling circle amplification (RCA), bridge PCR, picotiter PCR, emulsion PCR and reverse transcriptase PCR (RT-PCR). Other suitable amplification methods include the ligase chain reaction (LCR), loop-mediated isothermal amplification reaction (LAMP), recombinase polymerase reaction (RPA), transcription amplification, self-sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP -PCR), arbitrarily primed polymerase chain reaction (AP-PCR), degenerate oligonucleotide-primed PCR (DOP- PCR) and nucleic acid based sequence amplification (NABS A). Preferably, the amplification reaction is a qPCR method.

PCR-based assays can be used to detect the presence of the second nucleic acids in the sample displaced from their aptamers due to a target-protein and aptamer interaction. In such assays, one or more primers specific to a particular second nucleic acid are used for amplification. These primers have sequences specific to a particular second nucleic acid such that they only hybridize and initiate PCR when they are complementary to a second nucleic acid. If the second nucleic acid of interest is present in the sample and has been displaced from its aptamer, the primer will hybridize for an amplification reaction. To determine whether a particular second nucleic acid is available for amplification (indicating the target protein cognate to the aptamer is present sample), the PCR products may be detected to identify and quantitate the target protein in the sample. The PCR product can be detected, for example, by methods using an intercalating dye, like SybrGreen or ethidium bromide, hybridizing the PCR products to a solid substrate, such as a bead (e.g., magnetic or fluorescent beads, such as Luminex beads), or detecting them through an intermolecular reaction, such as FRET, or sequence-specific detection methods such as TaqMan probes, molecular beacons or scorpion probes.

The second nucleic acids may include a PCR handle that functions as a primer site used for subsequent PCR amplification. Accordingly, the inclusion of PCR-handle-specific primers during amplification of the released second nucleic acids. In certain aspects, the second nucleic acids comprise a universal primer sequence and are amplified using a universal primer. According to aspects of the present disclosure, the term “universal primer sequence” generally refers to a primer binding site, e.g., a primer sequence that would be expected to hybridize (base- pair) to, and prime, one or more loci of complementary sequence, if present, on any nucleic acid fragment. In some embodiments, the universal primer sequences used with respect to the present methods are P5 and P7.

In certain aspects, the amplified second nucleic acids are used to construct a library. Certain methods of the invention include, for example, counting second nucleic acids amplified in the library. For example and without limitation, counting may include any of the following: counting the total number of amplified targets; comparing the number of targets amplified to the number of different aptamers; comparing the absolute or relative abundance of different amplified targets; and counting the number of target products that arose from independent amplification of the same second nucleic acid molecule.

Methods of the disclosure may further include sequencing amplified second sequences in the library. Sequencing can be used, for example, to identifying and quantify the released second nucleic acids. In certain aspects, amplification products of the second sequences are sequenced for purposes of identification and/or quantification.

In certain aspects, the second nucleic acid includes one or more barcode regions. The term barcode region may comprise any number of barcodes, index or index sequence, UMIs, which are unique, i.e., distinguishable from other barcode, or index, UMI sequences. The sequences may be of any suitable length which is sufficient to distinguish the barcode, or index, sequence from other barcode sequences. A barcode, or index, sequence may have a length of 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides, or more. In some embodiments, the barcodes, or indices, are predefined and selected at random.

In some methods of the invention, a barcode may comprise unique molecular identifiers (UMIs). UMIs are a type of barcode that may be provided to a sample to make each nucleic acid molecule, together with its barcode, unique, or nearly unique. This may be accomplished by adding one or more UMIs to the second nucleic acids. By selecting an appropriate number of UMIs, every second nucleic acid molecule in the sample, together with its UMI, will be unique or nearly unique.

In certain aspects, UMIs can be used to quantify the number of individual released second nucleic acids, and by extension, the aptamers to which they were hybridized and the analytes (e.g., proteins) that displaced the second nucleic acids. As every second nucleic acid molecule in a sample together with its UMI or UMIs is unique or nearly unique, after amplification and sequencing, sequences with identical UMI sequences may be considered to refer to the same starting nucleic acid molecule. Thus, sequence data (e.g., sequence reads) with identical or near-identical UMIs can be de-duplicated. The de-duplicated sequence data represents quantity or number of unique starting second nucleic acids before amplification and sequencing.

UMIs are also advantageous in that they can be used to correct for errors created during amplification, such as amplification bias or incorrect base pairing during amplification. For example, when using UMIs, because every nucleic acid molecule in a sample together with its UMI or UMIs is unique or nearly unique, after amplification, molecules with identical sequences may be considered to refer to the same starting nucleic acid molecule, thereby reducing amplification bias.

The invention may include detecting changes in target proteins indicative of a disease or medical condition. The methods may include providing a diagnosis, prognosis, or course of treatment for a disease or medical condition.

In certain aspects, the aptamer is a single-stranded nucleic acid about 20 bases in length. In certain aspects, the aptamer is a single-stranded nucleic acid between 5-10 bases in length, 10- 20 bases in length, 20-30 bases in length, 30-40 bases in length, 40-50 bases in length, 50-60 bases in length, 60-70 bases in length, 70-80 bases in length, 80-90 bases in length, or 90-100 bases in length.

In certain aspects, the single-stranded nucleic acids of the aptamers are designed to exhibit kinetics favorable to disassociating in the presence of the target analyte. The aptamers may be designed using techniques such as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). In SELEX, a library of nucleic acids is prepared, with each having a target binding region composed of random nucleobases. The nucleic acids are contacted with a target protein. Nucleic acids bound to the target proteins are eluted, while unbound nucleic acids are discarded. The eluted nucleic acids are amplified. In certain methods, the eluted nucleic acids are contacted with proteins similar to the target protein. Nucleic acids that bind to these similar proteins are discarded. The remaining nucleic acids are sequenced and their K_d determined.

Those nucleic acids with an appropriate K_d are selected for use as aptamers.

In some aspects, methods and systems of the disclosure are used to detect and/or quantify one or more proteins present in a biological sample. In certain aspects, the sample may be derived from live cells, for example, a tissue or body fluid sample of a human or non-human animal subject. The sample may be obtained via a fine needle aspirate, a biopsy, or body fluid collection, such as an effluent or discharge fluid. In certain aspects, the sample includes or is derived from one or more of blood, plasma, cerebrospinal fluid, semen, fecal matter, saliva, urine, bile, bone marrow, tissue, mucus, tears, and sweat. In certain aspects, due to the sensitivity and specificity of the presently disclosed systems and methods, the sample is obtained by minimally-invasive means. In certain aspects, target proteins for detection are derived from circulating cells, cellular components, extracellular vesicles, exosomes, antigens, prokaryotic cells, fungi, viruses, and/or combinations thereof. The invention is useful for the detection of circulating tumor cells, fetal cellular material or any other material amenable to detection in a liquid biopsy format. In certain aspects, the protein is a viral protein, a bacterial protein, or other protein associated with a pathology. In certain aspects, the pathology is Alzheimer’s disease and the target protein is a p- tau protein. In certain aspects, the target protein is indicative of a disease, such as cancer. In certain aspects, the target protein contains a mutation or misfolding indicative of a genetic alterations, including substitutions, insertions, deletions, truncations, single nucleotide polymorphisms, changes in copy number, changes in expression, and the like.

In some methods of the invention, the initial binding construct of the second nucleic acid may be used to screen libraries of unknown aptamers to select the most appropriate combination of aptamer and second nucleic acid to favor the kinetics in the selection of aptamers used in the invention.

Methods of the disclosure may include any known sequencing technique to identify and/or quantify the second nucleic acids.

A sequencing technique that can be used in the methods of the invention, includes any of those commercialized by Illumina, such as those described in U.S. Pat. Nos. 5,750,341; 6,306,597; and 5,969,119, which are each incorporated by reference. In some such methods, directional (strand-specific) libraries are prepared, and the selected single-stranded nucleic acid is amplified, for example, by PCR. The resulting nucleic acid is then denatured and the single- stranded amplified polynucleotides are randomly attached to the inside surface of flow-cell channels. Unlabeled nucleotides are added to initiate solid-phase bridge amplification to produce dense clusters of double-stranded DNA. To initiate the first base sequencing cycle, four labeled reversible terminators, primers, and DNA polymerase are added. After laser excitation, fluorescence from each cluster on the flow cell is imaged. The identity of the first base for each cluster is then recorded. Cycles of sequencing are performed to determine the fragment sequence one base at a time.

In certain aspects, the methods of the present invention may employ sequencing by ligation methods commercialized by Applied Biosystems (e.g., SOLiD sequencing). In other embodiments, the methods of the present invention may employ sequencing by synthesis using the methods commercialized by 454/Roche Life Sciences, including but not limited to the methods and apparatus described in Margulies et al., Nature (2005) 437:376-380 (2005); and U.S. Pat. Nos. 7,244,559; 7,335,762; 7,211,390; 7,244,567; 7,264,929; and 7,323,305. In other embodiments, the methods of the present invention may employ the sequencing methods commercialized by Helicos BioSciences Corporation (Cambridge, Mass.), such as described in U.S. application Ser. No. 11/167,046, and U.S. Pat. Nos. 7,501,245; 7,491,498; 7,276,720; and in U.S. Patent Application Publication Nos. US20090061439; US20080087826; US20060286566; US20060024711; US20060024678; US20080213770; and US20080103058, which are each incorporated by reference. In other embodiments, the methods of the present invention may employ sequencing by the methods commercialized by Pacific Biosciences, such as Single Molecule Real-Time (SMRT) sequencing and as described in U.S. Pat. Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050; 7,462,468; 7,476,503; 7,315,019; 7,302,146; 7,313,308; and

US Application Publication Nos. US20090029385; US20090068655; US20090024331; and US20080206764, each of which is incorporated by reference.

Another example of a sequencing technique that can be used in the methods of the provided invention is nanopore sequencing (see e.g. Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001), which is incorporated by reference. A nanopore can be a small hole of the order of 1 nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential across it can result in a slight electrical current due to conduction of ions through the nanopore. The amount of current that flows is sensitive to the size of the nanopore. As a DNA or label molecule passes through a nanopore, each nucleotide on, or label from, the DNA molecule obstructs the nanopore to a different degree. Thus, the change in the current passing through the nanopore as the DNA molecule or label passes through the nanopore can represent a reading of the DNA sequence.

Another example of a sequencing technique that can be used in the methods of the invention is semiconductor sequencing, such as provided by Ion Torrent (e.g., using the Ion Personal Genome Machine (PGM)). Ion Torrent technology can use a semiconductor chip with multiple layers, e.g., a layer with micro-machined wells, an ion-sensitive layer, and an ion sensor layer. Nucleic acids can be introduced into the wells, e.g., a clonal population of single nucleic can be attached to a single bead, and the bead can be introduced into a well. To initiate sequencing of the nucleic acids on the beads, one type of deoxyribonucleotide (e.g., dATP, dCTP, dGTP, or dTTP) can be introduced into the wells. When one or more nucleotides are incorporated by DNA polymerase, protons (hydrogen ions) are released in the well, which can be detected by the ion sensor. The semiconductor chip can then be washed and the process can be repeated with a different deoxyribonucleotide. A plurality of nucleic acids can be sequenced in the wells of a semiconductor chip. The semiconductor chip can comprise chemical-sensitive field effect transistor (chemFET) arrays to sequence DNA (for example, as described in U.S. Patent Application Publication No. 2009/0026082), which is incorporated by reference. Incorporation of one or more triphosphates into a new nucleic acid strand at the 3' end of the sequencing primer can be detected by a change in current by a chemFET. An array can have multiple chemFET sensors.

Sequencing generally produces sequencing data, such as sequence reads, that can be used to identify and quantify the released second nucleic acids. Sequencing produces sequence data that can be assembled or analyzed in accordance with known methods, such as those discussed in U.S. Patent No. 8,209,130, incorporated by reference.

EXAMPLES

Example 1 - Detecting SARS-CoV-2 Spike Protein with Nucleic Acid Aptamer

The present Inventors performed a proof-of-principle experiment that shows the presently disclosed systems and methods are able to accurately detect the presence of the SARS-CoV-2 viral proteins when present in a sample at attomolar concentrations.

Fig. 2 provides a schematic of the experiment. The single-stranded nucleic acid aptamers (Cv2 A) were hybridized to a release strand of nucleic acid and were anchored to a solid surface via a biotin/streptavidin interaction. The aptamer included a sequence with sites that bind to the SARS-CoV-2 spike protein.

The SARS-CoV-2 spike protein was added to a solution containing the aptamers at different concentrations. The aptamers bound the spike proteins in the samples, displacing the second nucleic acid (Cv2 comp) from the aptamers.

The solid surface was washed to remove the released second nucleic acid, which was collected and amplified to detect the presence and quantity of the spike protein in each sample. The amplified products were collected, fluorescently labeled, and run on gels in a 10% Native PAGE assay at 170 V for 35 minutes.

Fig. 3A shows the structure of the SARS-CoV-2 spike protein, with its receptor binding domain (RBD) highlighted. The nucleic acid aptamer (2019nCoV-RBD-lC) is shown interacting with the RBD of the spike protein.

Fig. 3B provides a closeup view of the protein-aptamer interaction. As shown, several amino acids in the protein RBD interact with and/or form bonds with bases in the nucleic acid aptamer.

Fig. 4 provides pictures of the gels, each gel representing a separate repetition of the experiment. The lanes of the gels each included:

Lane 1 100 base pair DNA ladder

Lane 2 Amplified products from positive control at 1 mM

Lane 3 Amplified products from SAR.S-CoV-2 spike protein at 4 pM

Lane 4 Amplified products from SAR.S-CoV-2 spike protein at 400 fM

Lane 5 Amplified products from SAR.S-CoV-2 spike protein at 40 fM

Lane 6 Amplified products from SAR.S-CoV-2 spike protein at 4 fM

Lane 7 Amplified products from SAR.S-CoV-2 spike protein at 400 aM

Lane 8 Amplified products from negative control

Lane 9 Primers

In this experiment, the positive control (Lane 2) was the second nucleic acid free from aptamers. As expected, the bright band at about 100 base pairs reveals that these second nucleic acids were available for amplification. The negative control, Lane 8, was from an assay run in which the aptamer was attached to the solid surface, but not hybridized to a second nucleic acid. The lack of the 100 base pair band in lane 8 confirms that the second nucleic acid was not in the sample and available for amplification.

Lanes 2-7 of each gel contain the amplification products of the second nucleic acids after contacting the aptamers with SAR.S-CoV-2 spike proteins at various concentrations. The lowest concentration of spike protein tested was at 400 aM (Lane 7). As shown by the band at around 100 base pairs in each of these lanes, the second nucleic acid was successfully and specifically displaced and amplified despite the miniscule concentrations of spike protein. The amount of spike protein in the lowest concentration aliquots (400 aM) was quantified, which provided preliminary results of approximately 1,200 molecules of spike protein detected in the sample. The spike protein of SARS-CoV-2 is a trimer, thus the 1,200 molecules quantified represent the equivalent of 400 trimers. Given the average number of turners per virus particle, the presently disclosed method was able to quantify an amount of spike protein equivalent to only about 16 viral particles.

Thus, the presently-disclosed methods are able to detect and quantify viral proteins with high specificity at an unprecedently low concentration.

Fig. 5 provides a picture of a gel showing results from separate repetitions of the experiment. In this experiment, the positive controls (Lane 3 and 10) are the second nucleic acid free from aptamers. The bright band at about 100 base pairs reveals that the released second nucleic acids were available for amplification. The negative control, Lane 7, was from an assay run in which the aptamer was attached to the solid surface and hybridized to its complementary strand and was contacted with a vehicle solution without target protein present. The lack of the 100 base pair band in lane 7 confirms that the second nucleic acid was not in the sample and available for amplification.

Lanes 4-6 of the gel contain the amplification products of the second nucleic acids after contacting the aptamers with SARS-CoV-2 spike proteins at various concentrations. The lowest concentration of spike protein that registered a detection was at 40 aM (Lane 5), as shown by the band at around 100 base pairs in the lane.

Example 2 - Nucleic Acid Aptamer Displaced by Protein

The present Inventors performed an experiment showing that the second nucleic acid is displaced from the aptamer by the SARS-CoV-2 spike protein as expected.

Fig. 6 provides an overview of the experiment 601. An aptamer 603 was prepared and anchored to a solid surface 607. The second nucleic acid used in Example 1 was, in essence, split apart to form nucleic acids PI and P2. PI was decorated with a Cy5 fluorophore and P2 with a Cy3 fluorophore. P2 and P3 hybridized to the aptamer, including at a location on the aptamer that interacts with the spike protein. When PI and P2 hybridized to the aptamer, the Cy5 and Cy3 fluorophores were disposed from one another at a distance to cause a Forster resonance energy transfer interaction between the fluorophores. The aptamer was then contacted with the spike protein.

Fig. 7 shows an emission spectrum for repetitions of the experiment 601. In the first, the spike protein was provided at a concentration of 83 nM and in the second at 1 mM. On the spectrums, the dark line represents the spectrum taken after contacting the aptamers with the spike protein. The increase in emission at approximately 650 nm indicates that the Cy3 of P2 was removed from its proximity to the Cy5 of PI. This indicates that the PI and P2 displaced. Further, as P2 hybridized to the aptamer at a location suspected of binding to the spike protein, it is clear that PI and P2 were displaced due to the aptamer-protein interaction.

Example 3 - Predicting the Progression of Alzheimer’s Disease

The progression of Alzheimer’s Diseases (AD) is associated with the small increase of circulating free phosphorylated tau (p-tau) protein within cerebral spinal fluid (CSF). In dominantly inherited Alzheimer’s disease, increases in various p-tau variants, such as p-taul81, can be detected in CSF up to 15 years before symptom onset. However, the lumbar puncture procedure necessary to detect p-tau levels in CSF is both expensive and painful, and is thus not suited for population-level screening in asymptomatic individuals. Assays to detect p-taul81 in plasma represent a potential alternative to CSF-based screening, but levels of p-taul81 in the blood are very low, and to date there are no clinically approved assays for p-taul81 in blood.

The presently disclosed systems and methods can be used to predict the progression or onset of AD in a subject. In such a method, a plasma sample is obtained from a subject. Target- specific, single-stranded nucleic acid aptamers, each hybridized to a second nucleic acid, are introduced into the sample. The aptamers have a nucleic acid sequence that interacts with one of p-taul81, p-tau202, and p-tau441. When an aptamer captures a cognate p-tau protein, the second nucleic acid is displaced. The released second nucleic acids are collected and amplified to identify and quantify p-tau isoforms in the sample. The quantity of p-tau isoforms in the sample is used to predict the progression or onset of AD in the subject. The p-tau isoforms are detected at an LoD of about 0.002 pg/mL (40aM) and quantified at a FLoQ of about 0.1 pg/mL (2fM). INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

EQUIVALENTS

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

CLAIMS What is claimed is:

1. A method for detecting an analyte in a sample, the method comprising: contacting a biological sample with a target-specific single-stranded nucleic acid aptamer hybridized to a second nucleic acid with regions complementary to the aptamer; wherein, upon binding of a target analyte to said aptamer, the second nucleic acid is released from the aptamer; and detecting the released second nucleic acid as indicative of the presence of the analyte.

2. The method of claim 1, wherein the analyte is selected from the group consisting of proteins, metabolites, peptide fragments, and antibodies.

3. The method of claim 1, wherein said second nucleic acid comprises a UMI.

4. The method of claim 2, wherein the protein is identifiable in the sample at or below a concentration of about 1 fM.

5. The method of claim 1, wherein the detecting step comprises amplifying the released second nucleic acid.

6. The method of claim 5, wherein said amplifying step is qPCR.

7. The method of claim 6, wherein the amount of the target protein is quantifiable in the sample at or below a concentration of about 10 fM.

8. The method of claim 1, wherein the sample is contacted with a plurality of different aptamers, each hybridized to a different second nucleic acid.

9. The method of claim 8, wherein each different target-specific aptamer captures a different target analyte.

10. The method of claim 1, wherein the aptamer is attached to a solid surface.

11. The method of claim 10, wherein the aptamer is attached to via a biotin/streptavidin interaction.

12. The method of claim 2, wherein the protein is a protein associated with a pathology.

13. The method of claim 12, wherein the pathology is a viral infection.

14. The method of claim 13, wherein the viral infection is a SARS infection.

15. The method of claim 14, wherein the protein is a viral spike protein

16. The method of claim 13, wherein the protein is a viral spike protein.

17. The method of claim 12, wherein the sample is contacted with a plurality of different target-specific aptamers that are each hybridized to a different second nucleic acid, wherein each different target-specific aptamer captures a different isoform or variant of the protein.

18. The method of claim 12, wherein the pathology is Alzheimer’s Disease and the target protein is a p-Tau protein.

19. The method of claim 1, wherein the sample from the subject is obtained from one or more of blood, plasma, cerebrospinal fluid, semen, fecal matter, saliva, urine, bile, bone marrow, tissue, mucus, tears, and sweat.

20. A method for selecting aptamers for detection of an analyte in a biological sample, the method comprising the steps of preparing a library of aptamers comprising second nucleic acids that hybridized thereto; exposing said library to a plurality of known analytes, at least one of which is capable of binding to one or more of said aptamers; and determining aptamers bound by a member of said plurality by detection of released second nucleic acid, thereby to select aptamers for detection of said analyte.