KR20240124818A - Design platform of bi-specific nucleic acid molecules - Google Patents

Abstract

The present invention relates to a platform for designing a dual target nucleic acid molecule that targets two genes and simultaneously suppresses their expression. In the present invention, by introducing mismatches at specific nucleotide positions of the dual target nucleic acid molecule, the off-target problem and the bias of the gene expression suppression effect of conventional dual target nucleic acid molecules are improved, and therefore, this can be utilized in the design of a dual target nucleic acid molecule.

Description

{Design platform of bi-specific nucleic acid molecules}

The present invention relates to a platform for designing dual target nucleic acid molecules that simultaneously target and suppress the expression of two genes.

Technology that suppresses gene expression is an important tool in the development of therapeutic agents and target verification for disease treatment. Since its role was discovered, RNA interference (RNAi) has been found to act on sequence-specific mRNA in various types of mammalian cells (Silence of the transcripts: RNA interference in medicine. J Mol Med (2005) 83: 764-773). RNAi is a phenomenon in which short interfering RNA (siRNA), a double-helix structured small interfering ribonucleic acid of 21-25 nucleotides in size, specifically binds to a transcript (mRNA transcript) with a complementary sequence and degrades the transcript, thereby suppressing the expression of a specific protein. Inside the cell, RNA duplexes are processed by an endonuclease called Dicer and converted into 21 to 23-base pair (bp) siRNA, which binds to RISC (RNA-induced silencing complex) and sequence-specifically inhibits the expression of target genes through a process in which the guide (antisense) strand recognizes and degrades the target mRNA (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002. 1, 503-514). According to Bertrand's research team, siRNA for the same target gene has a more excellent effect of inhibiting mRNA expression in vitro and in vivo than antisense oligonucleotides (ASO), and the effect lasts a long time (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004). The RNAi technology-based therapeutics market including siRNA is expected to form a global market size of more than 12 trillion won by 2020, and the target to which the technology can be applied has expanded dramatically, so it is evaluated as a next-generation gene therapy technology that can treat diseases that are difficult to treat with existing antibody and compound-based drugs. In addition, since the mechanism of action of siRNA is to complementarily bind to the target mRNA and regulate the expression of the target gene in a sequence-specific manner, compared to the long development period and development cost required for existing antibody-based drugs or chemical substances (small molecule drugs) until they are optimized for a specific protein target, the applicable targets can be dramatically expanded, the development period is shortened, and lead compounds optimized for all protein targets, including target substances that cannot be pharmaceutically viable, have the advantage (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13(4):664-670). Accordingly, research is being conducted to utilize this ribonucleic acid-mediated interference phenomenon to develop various disease treatments, especially tumor treatments, by selectively inhibiting the expression of specific proteins at the transcriptome level, as it suggests a solution to the problems occurring in the development of existing chemical synthetic drugs. In addition, siRNA therapeutics have the advantage of having a clear target and predictable side effects, unlike existing anticancer drugs. However, this target specificity can be a cause of low therapeutic efficacy in the case of tumors, which are diseases caused by problems with various genes. In addition, in the case of tumors, cancer cannot be treated by controlling a single gene, and anticancer drug resistance often occurs. Therefore, it is difficult to control cancer by targeting only a single gene when treating cancer with gene therapy. To solve this problem, if siRNAs for multiple genes are produced and introduced individually, the number that can be delivered as a vector is limited and the off-target effect increases, making it difficult to achieve the intended effect. Therefore, although sequence design is necessary so that a single nucleic acid sequence can simultaneously target multiple genes related to the intended disease, it is very difficult to design dual-target nucleic acid molecules due to sequence diversity, the possibility of off-targets, etc.

Currently, shRNA or miRNA generated from bicistronic sequences encoded as RNAi tools are used to suppress two genes, but in many cases, there has been a problem in that relatively long transcripts are generated and the desired structure is not formed. In addition, there is a method of using multiple promoters to encode separate shRNAs under independent promoters, but this has a problem in that biased production of shRNAs occurs, such that only one of the two targeted genes is down-regulated. Therefore, it is necessary to develop a next-generation dual targeting method that solves the off-target problem caused by the unbalanced down-regulation of two different genes.

Currently, as a method for designing a dual-specific nucleic acid molecule, there is a method disclosed in Republic of Korea Application No. 10-2019-0160720. The method can design a nucleic acid molecule targeting the mRNA of different genes, but 1) there is a problem that a blank region may be derived during the segmentation process by segmenting the DNA sequence of a specific gene at a specific interval to obtain sequence information and that the design efficiency may be low due to the presence of an intron region in the DNA sequence. In addition, 2) in the case of each sequence of a dual-specific nucleic acid molecule, mismatching is mostly allowed, but the off-target effect and inhibition efficiency differ significantly depending on the location of the mismatch, so even if a dual-specific nucleic acid molecule is designed, there is a problem that the designed dual-specific nucleic acid molecule may have an off-target effect or a low inhibition efficiency.

Accordingly, the inventors of the present invention, in order to solve the above problems, 1) increased the efficiency of designing a dual-specific nucleic acid molecule sequence by using an mRNA sequence rather than a DNA sequence of a gene when designing a dual-specific nucleic acid molecule, and 2) completed the present invention by deriving a mismatch position capable of maximizing the effect of a dual-specific nucleic acid molecule.

Accordingly, it is an object of the present invention to provide a dual target nucleic acid molecule comprising a mismatch at a specific position.

In addition, it is an object of the present invention to provide a method for designing a dual target nucleic acid molecule.

To achieve the above purpose, the present invention provides a dual target nucleic acid molecule containing a mismatch at a specific nucleotide position.

In addition, the present invention provides a method for designing a dual target nucleic acid molecule without off-target effects.

In the present invention, the off-target problem and the bias of the gene expression inhibition effect of conventional dual target nucleic acid molecules are improved by introducing a mismatch at a specific nucleotide position of the dual target nucleic acid molecule, and thus this can be utilized in the design of dual target nucleic acid molecules.

Figure 1 is a diagram illustrating the principle of the algorithm for deriving dual target siRNA candidates.
Figure 2 is a diagram showing the difference between a conventionally designed dual target siRNA pair and a dual target siRNA pair designed to include a mismatch in the present invention.
Figure 3 is a diagram analyzing the mismatch positions of the designed siRNA pair and the corresponding gene expression inhibition efficiency of each strand of the siRNA pair:
a: Position of mismatch in siRNA pair at full sequence length 1; and
b: Mismatch location and gene expression inhibition efficiency (KD _eff ).
Figure 4 is a diagram showing the location of mismatches for optimized gene expression suppression efficiency in dual target siRNA, based on a single-strand siRNA.
Figure 5 is a diagram confirming the selectivity of siRNA gene expression inhibition efficiency according to mismatch position and structure.
FIG. 6 is a block diagram of a device (200) for designing a dual target nucleic acid molecule according to one embodiment.

Hereinafter, the present invention will be described in detail with examples of the present invention. However, the following examples of the present invention are presented as examples of the present invention, and the present invention is not limited thereby, and the present invention can be variously modified and applied within the scope of the following claims and equivalents interpreted therefrom.

Unless otherwise indicated, nucleic acids are written in a 5' to 3' orientation from left to right. Numerical ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein.

The terms used in the present invention are selected from the most widely used general terms possible while considering the functions of the present invention, but this may vary depending on the intention of engineers working in the relevant field, precedents, the emergence of new technologies, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meanings thereof will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the overall contents of the present invention, rather than simply the names of the terms.

When a part of the specification is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated. In addition, terms such as "part", "module", etc., used in the specification mean a unit that processes at least one function or operation, which may be implemented by hardware or software, or a combination of hardware and software.

In one aspect, the present invention relates to a dual target nucleic acid molecule comprising 1) a first nucleic acid molecule targeting mRNA of a first gene; and 2) a second nucleic acid molecule targeting mRNA of a second gene, wherein the first nucleic acid molecule and the second nucleic acid molecule are partially complementary, and a base pair of the first nucleic acid molecule and the second nucleic acid molecule is mismatched at a position corresponding to 0.25 to 0.40, or a position corresponding to 0.60 to 0.75 from the 5' end or the 3' end of the first nucleic acid molecule or the second nucleic acid molecule with respect to a total nucleotide (nt) length of 1.00 of the first nucleic acid molecule or the second nucleic acid molecule.

In one embodiment of the present invention, a base pair between the first nucleic acid molecule and the second nucleic acid molecule may be mismatched at a position corresponding to 0.25 to 0.30, or a position corresponding to 0.70 to 0.75 from the 5' end or the 3' end of the first nucleic acid molecule or the second nucleic acid molecule with respect to a total nucleotide (nt) length of 1.00 of the first nucleic acid molecule or the second nucleic acid molecule.

In one embodiment, the first nucleic acid molecule can have a sequence that is 100% complementary to an mRNA of the first gene, and the second nucleic acid molecule can have a sequence that is 100% complementary to an mRNA of the second gene.

In one embodiment, the first nucleic acid molecule and the second nucleic acid molecule may be mismatched by 1 to 3 base pairs.

In one embodiment, the first nucleic acid molecule or the second nucleic acid molecule can be 14 to 30 mer, more preferably 17 to 24 mer.

In one embodiment, the dual target nucleic acid molecule can be a double stranded (ds) small interfering RNA (siRNA) or shRNA.

In one embodiment, when the dual target nucleic acid molecule is 20mer, 1) the base at the 7th position from the 5' end of the first nucleic acid molecule and the base at the 14th position from the 5' end of the second nucleic acid molecule may be mismatched; 2) the base at the 9th position from the 5' end of the first nucleic acid molecule and the base at the 12th position from the 5' end of the second nucleic acid molecule may be mismatched; and 3) the base at the 14th position from the 5' end of the first nucleic acid molecule and the base at the 7th position from the 5' end of the second nucleic acid molecule may be mismatched.

In one embodiment, the nucleic acid molecule may be a double-stranded siRNA in which a first nucleic acid molecule, a single-stranded siRNA, targeting the mRNA of the first gene, and a second nucleic acid molecule, a single-stranded siRNA, targeting the mRNA of the second gene, partially complementarily bind to each other, and the sense strand (guide RNA) of the siRNA consisting of a double strand (ds) may be an siRNA that complementarily binds to the mRNA of the first gene, and the antisense strand (passenger RNA) of the siRNA may be an siRNA that complementarily binds to the mRNA of the second gene.

In one embodiment, the first nucleic acid molecule can inhibit expression of a first gene by RNA interference, and the second nucleic acid molecule can inhibit expression of a second gene by RNA interference, so that the nucleic acid molecules of the present invention can simultaneously inhibit expression of two genes.

In one embodiment, the nucleic acid of the siRNA may comprise a nucleotide analogue in which the sugar or backbone of one or more nucleotides is modified.

In one embodiment, the modification of the sugar may be such that the hydroxyl group (-OH) at the 2' carbon position of ribose is substituted with a methyl group (-CH ₃ ) (OMe), a methoxy group (-OCH ₃ ), an amine group (-NH ₂ ), fluorine (-F) (fluoro), O-2-methoxyethyl group (MOE), O-propyl group, O-2-methylthioethyl group, O-3-aminopropyl group, O-3-dimethylaminopropyl group, ON-methylacetamido group or O-dimethylamidooxyethyl group, and it is more preferable that the 2'-hydroxyl group of ribose is substituted with 2'-MOE (2'- O -methoxyethyl), 2'-OMe (2'- O -Methyl) or 2'-F (fluoro).

In one embodiment, the modification of the backbone may be such that the phosphate backbone of the nucleotide is modified with phosphorothioate, phosphorodithioate, methyl phosphonate, alkylphosphonate, phosphoroamidate or boranophosphate, more preferably with phosphorothioate.

The term "reverse complementary sequence" or "reverse complementary sequence" used in the present invention means a sequence in the opposite direction of a gene sequence indicated from 5' to 3', indicated from 5' to 3'. More specifically, a general method of indicating a gene sequence is to indicate only one strand of a double strand of a gene in the 5' to 3' direction from the promoter position, and when the gene is transcribed, the complementary sequence of the gene indicated in the 5' to 3' direction, that is, the 3' to 5' sequence, is used as a template, and as a result, the 5' to 3' sequence from the promoter position becomes a sequence that matches the sequence of an mRNA transcript (in the case of an mRNA sequence, one strand). At this time, the sequence identical to the mRNA is called the sense sequence, and the sequence complementary to the sense sequence is called the antisense sequence. At this time, the sequence of the strand used as a template indicated in the 5' to 3' direction is called the reverse complementary sequence or reverse complementary sequence. That is, when the gene sequence is indicated as 5'-ATGCATGC-3', its reverse complement sequence is 5'-GCATGCAT-3'. Also, if the gene sequence is 5'-ATGCATGC-3', the sequence transcribed into mRNA is 5'-GCAUGCAU-3', and the antisense sequence of the transcribed mRNA is 5'-AUGCAUGC-3'.

The term "nucleic acid molecule" as used in the present invention refers to deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form, and includes natural nucleic acid analogs unless specifically stated otherwise (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

The term "expression suppression" as used in the present invention means causing a decrease in the expression (into mRNA) or translation (into protein) of a target gene, and preferably means that the target gene expression becomes undetectable or exists at an insignificant level.

The term "siRNA (small interfering RNA)" used in the present invention refers to a short double-stranded RNA that can induce RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. In general, siRNA is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. However, the dual target siRNA of the present invention has a sense RNA strand that is an antisense strand for a first gene and an antisense RNA strand that is an antisense strand for a second gene, so that the double-stranded siRNA can simultaneously suppress the expression of the first gene and the second gene, and is therefore provided as an efficient gene knock-down method or as a gene therapy method.

The term "shRNA (short hairpin RNA)" used in the present invention refers to RNA which partially includes a palindromic base sequence in a single-stranded RNA, thereby forming a double-stranded structure in the 3' region and a hairpin-like structure, and which can be cleaved by dicer, a type of RNase present in the cell after being expressed in the cell and converted into siRNA. The length of the double-stranded structure is not particularly limited, but is preferably 10 nucleotides or more, and more preferably 20 nucleotides or more. In the present invention, the shRNA can be included in an expression cassette, and the shRNA can be produced by constructing an expression cassette encoding shRNA by sequentially linking TTGGATCCAA (TTGGATCCAA loop) or TTCAAGAGAG (TTCAAGAGAG loop) to the 3' of the sense strand, the antisense strand, and TT in this manner and expressing the same in a cell.

In one embodiment, the base sequence targeting the first gene can include a base sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity to the reverse complement sequence of the base sequence targeting the second gene, and the base sequence targeting the second gene can include a base sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity to the reverse complement sequence of the base sequence targeting the first gene.

Variants of the base sequence targeting the first gene or the base sequence targeting the second gene are included within the scope of the present invention. The expression cassette of the present invention is a concept including functional equivalents of the nucleic acid molecule constituting it, for example, variants in which a part of the base sequence of the nucleic acid molecule is modified by deletion, substitution or insertion, but which can perform the same function functionally as the base sequence molecule. The "% sequence homology" for a nucleic acid molecule is determined by comparing two optimally aligned sequences with a comparison region, and a part of the nucleic acid molecule sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

In one aspect, the present invention relates to a recombinant expression vector expressing a nucleic acid molecule of the present invention.

The recombinant vector of the present invention can be produced by a recombinant DNA method known in the art.

Nonviral vectors useful for delivering the dual target nucleic acid molecules in the present invention include all vectors commonly used in gene therapy, for example, various plasmids and liposomes capable of expression in eukaryotic cells.

In order for the dual target nucleic acid molecule siRNA of the present invention to be properly transcribed in the delivered cell, it is preferable that at least the base sequence encoding the shRNA including it be operably linked to a promoter. Any promoter that can function in a eukaryotic cell may be used as the promoter. In order to efficiently transcribe the dual target nucleic acid molecule siRNA or shRNA, a regulatory sequence including a leader sequence, a polyadenylation sequence, a promoter, an enhancer, an upstream activating sequence, a signal peptide sequence, and a transcription terminator may be additionally included as needed.

Viruses or viral vectors useful for delivering the dual target nucleic acid molecule in the present invention include, but are not limited to, baculoviridiae, parvoviridiae, picornoviridiae, herepesviridiae, poxviridiae, adenoviridiae, and the like.

In one aspect, the present invention relates to a method for designing a dual target nucleic acid molecule, comprising the step of selecting, from a database of pre-formed dual target nucleic acid molecules, a base pair mismatch between a first nucleic acid molecule and a second nucleic acid molecule at a position corresponding to 0.25 to 0.40, or a position corresponding to 0.60 to 0.75 from a 5' or 3' end of the first nucleic acid molecule or the second nucleic acid molecule, with respect to a total nucleotide (nt) length of 1.00 of the first nucleic acid molecule or the second nucleic acid molecule of the dual target nucleic acid molecule.

In one embodiment, a base pair mismatch between the first nucleic acid molecule and the second nucleic acid molecule may occur at a position that is 0.25 to 0.30, or a position that is 0.70 to 0.75 from the 5' end or the 3' end of the first nucleic acid molecule or the second nucleic acid molecule with respect to a total nucleotide (nt) length of 1.00 of the first nucleic acid molecule or the second nucleic acid molecule.

In one embodiment, the first nucleic acid molecule can have a sequence that is 100% complementary to an mRNA of the first gene, and the second nucleic acid molecule can have a sequence that is 100% complementary to an mRNA of the second gene.

In one embodiment, the first nucleic acid molecule and the second nucleic acid molecule may be mismatched by 1 to 3 base pairs.

In one embodiment, the first nucleic acid molecule or the second nucleic acid molecule can be 14 to 30 mer, more preferably 17 to 24 mer.

In one embodiment, the dual target nucleic acid molecule can be a double stranded (ds) small interfering RNA (siRNA) or shRNA.

In one embodiment, when the length of the dual target nucleic acid molecule is 20mer, 1) the base at the 7th position from the 5' end of the first nucleic acid molecule and the base at the 14th position from the 5' end of the second nucleic acid molecule may be mismatched; 2) the base at the 9th position from the 5' end of the first nucleic acid molecule and the base at the 12th position from the 5' end of the second nucleic acid molecule may be mismatched; and 3) the base at the 14th position from the 5' end of the first nucleic acid molecule and the base at the 7th position from the 5' end of the second nucleic acid molecule may be mismatched.

In one embodiment, the generation of the dual target nucleic acid molecule database can generate sequence information through a segmentation step of segmenting sequence information from the 5' end of the mRNA of the first gene into segments of 15 to 30 mer in length, and the segmentation step can be a multiple segmentation of 15 to 30 mer in length, starting from a point sequentially spaced apart by 1 nt (nucleotide) in the 3' end from the 5' end of the mRNA.

In one embodiment, the generation of the dual target nucleic acid molecule database can generate sequence information through a segmentation step of segmenting sequence information from the 3' end of the mRNA of the second gene into segments of 15 to 30 mer in length, and the segmentation step can be a multiple number of segmentations of 15 to 30 mer in length, starting from points sequentially spaced apart by 1 nt in the 5' end direction from the 3' end of the mRNA.

In one embodiment, the sequence information generated in the segmentation step may be generated in a length of 17 to 21 mer.

In one embodiment, the generation of the dual target nucleic acid molecule database can be generated by the design method of a dual target nucleic acid molecule of the reference document, Republic of Korea Patent Application No. 10-2019-0160720.

In addition, in one embodiment, (1) the sequence information can be generated through a segmentation step of segmenting the sequence information from the 5' end of the mRNA of the first gene into segments of 15 to 30 mer in length, and the segmentation step can be a multiple segmentation of 15 to 30 mer in length, starting from a point sequentially spaced apart by 1 nt (nucleotide) in the 3' end direction from the 5' end of the mRNA;

(2) The sequence information can be generated through a segmentation step that segments the sequence information from the 3' end of the mRNA of the second gene into 15 to 30 mer lengths to generate the sequence information, and the segmentation step can be segmenting multiple times into 15 to 30 mer lengths starting from a point sequentially spaced by 1 nt in the 5' end direction from the 3' end of the mRNA; and

(3) It may include a step of aligning sequence information generated from mRNA of the first gene and sequence information generated from mRNA of the second gene.

In one implementation, the step of scoring the matched sequences may be additionally included after the step of aligning.

In one embodiment, the scoring step is performed when the segmented sequence 1) is not located within 75 nt from a start codon, 2) the content of G base and C base is 36% to 52% with respect to the total number of bases, 3) the GC repeat sequence is less than 3, 4) the AT repeat sequence is less than 4, 5) the segmented sequence is a sense sequence, and a G base or a C base is present at the first position, 6) the segmented sequence is a sense sequence, and an A base is present at the third position, 7) the segmented sequence is a sense sequence, and a T or U base is present at the 10th position, 8) the segmented sequence is a sense sequence, and a G base is not present at the 13th position, 9) the segmented sequence is a sense sequence, and an A base is present at the 19th position, 10) the segmented sequence is a sense sequence. A weighting of 1 point may be given to the following cases: 1) if there is no G base or C base at the 19th position, 11) if the segmented sequence is an antisense sequence, and there is no A base, T base, or U base at the first position, 12) if the segmented sequence is an antisense sequence, and there is a G base at the first position of the sequence, 13) if the segmented sequence is an antisense sequence, and there is A at the 6th position, 14) if the segmented sequence is an antisense sequence, and the 19th sequence is not a G base or C base, 15) if the segmented sequence is an antisense sequence, and the 19th sequence is a U base or T base, and 16) if the segmented sequence is located in a coding sequence (CDS).

In one embodiment, the scoring step may be to weight a mismatch at a position corresponding to 0.25 to 0.40, or a position corresponding to 0.60 to 0.75 from the 5' end or the 3' end of the segmented sequence or the sequence matched with the segmented sequence, with respect to a total nucleotide (nt) length of 1.00 of the segmented sequence and the sequence matched with the segmented sequence.

Preferably, a weight may be given when a mismatch exists at a position corresponding to 0.25 to 0.30 or a position corresponding to 0.70 to 0.75 from the 5' end or the 3' end of the sequence matching the segmented sequence.

If a mismatch exists at a specific position in the sequence matched with the segmented sequence, the segmented sequence and the "matched sequence" can eliminate the off-target effect on genes other than the target gene to be targeted, respectively. The term "off-target effect" used in the present invention means the effect of targeting a gene other than the target gene to be targeted.

In one embodiment, after the scoring step, the method may further include a step of arranging the scores of the scoring from high to low. In addition, after the scoring step, the method may further include a step of aligning each of the segmented sequences and the sequences matched by the segmented sequences with a sequence of a database for previously formed gene transcripts, and then selecting each of the segmented sequences and the sequences matched by the segmented sequences as dual target nucleic acid molecules if there is no gene transcript matching each other except for the transcript of the gene from which it is derived.

In one embodiment, the alignment may use a striped smith-waterman algorithm, a needleman-wunsch algorithm, a levenshtein distance algorithm, a heuristic algorithm, or a hamming distance algorithm, and preferably, a striped smith-waterman algorithm may be used, but is not limited thereto. In addition, in the step, if the gene from which the aligned and matched sequences are derived (referred to as a second gene) is related to the same disease as the first gene, only the matched sequences may be selected, and the subsequent step may be performed.

In one embodiment, the aligning step may utilize a complementary sequence of the segmented sequence.

More specifically, in one embodiment of the present invention, (1) a sequence complementary to a sequence segmented in the 5' to 3' direction of the mRNA of the first gene and (2) a sequence segmented in the 3' to 5' direction of the mRNA of the second gene can be aligned with each other. For example, if the sequence of (1) is 5'-AUGAU-3', and the sequence complementary to the sequence of (1) can be 3'-UACUA-5', and the sequence of (2) can match 3'-UACUG-5' (mismatching A and G at 5'), and the sequence obtains a score exceeding a predetermined standard, it becomes a candidate sequence of a dual target nucleic acid molecule, and when designing siRNA or shRNA, the sequence targeting the mRNA of the first gene can be modified to 5'-AUCAU-3', and the sequence targeting the mRNA of the second gene can be modified to 3'-CAGUA-5', i.e., 5'-AUGAC-3'. That is, the final derived sequence can be 5'-AUCAU-3' (referred to as the first sequence) as a sequence targeting the mRNA of the first gene, and 5'-AUGAC-3' (referred to as the second sequence) as a sequence targeting the mRNA of the second gene. That is, in fact, the two sequences can have the potential to act as dual target nucleic acid molecules by partially complementary binding to each other, such that the A at the 5' end of the first sequence and the C at the 3' end of the second sequence are mismatched.

In addition, in another embodiment of the present invention, the complementary sequences of (1) a sequence segmented in the 5' to 3' direction of the mRNA of the first gene and (2) a sequence segmented in the 3' to 5' direction of the mRNA of the second gene can be aligned. For example, when the sequence of (1) is 5'-AUGAU-3' and the sequence of (2) is 3'-UACUG-5', its complementary sequence is 5'-AUGAC-3', and the above sequences can match each other (mismatching with U and C at 3'), and when the above sequences secure a score exceeding a predetermined standard, the above sequences become candidate sequences of a dual target nucleic acid molecule, so that when designing siRNA or shRNA, the sequence targeting the mRNA of the first gene can be modified to 5'-AUCAU-3', and the sequence targeting the mRNA of the second gene can be 5'-AUGAC-3'. That is, the final derived sequence can be 5'-AUCAU-3' (referred to as the first sequence) as a sequence targeting the mRNA of the first gene, and 5'-AUGAC-3' (referred to as the second sequence) as a sequence targeting the mRNA of the second gene. That is, in fact, the two sequences can have the potential to act as dual target nucleic acid molecules by partially complementary binding to each other, such that the A at the 5' end of the first sequence and the C at the 3' end of the second sequence are mismatched.

As used herein, the terms “sense sequence”, “segmented sequence” or “guide RNA” of siRNA may mean a sequence complementary to a 5’ to 3’ sequence of mRNA expressed from a first gene to be targeted (3’ -> 5’), and “antisense sequence” or “passenger RNA” of siRNA may mean a sequence complementary to a 3’ to 5’ sequence of mRNA expressed from a second gene to be targeted (5’ -> 3’).

The term "coding sequence (CDS)" used in the present invention may mean a sequence that is translated into a protein.

The term "start codon" as used in the present invention means the starting site at which the mRNA of the gene from which the sequences are derived is transcribed. For example, the requirement that the sequences are not located 75 bp from the start codon means that the segmented sequence is not derived from a sequence located within 75 bp of the start codon of the first gene from which the segmented sequence is derived.

In the present invention, when performing alignment, the sequence used is the 5' to 3' sequence direction sequence of the segmented sequence (antisense sequence for mRNA of the first gene), and in the case of the "matched sequence", the 5' to 3' sequence (antisense for mRNA of the second gene) can also be used. More specifically, the segmented sequence is 5'-ATGCTAC-3', and the sequence matched thereto is 5'-GTAGCAT-3', so that the segmented sequence targets the mRNA of the first gene, and the matched sequence targets the mRNA of the second gene. At this time, when performing alignment, the segmented sequence 5'-ATGCTAC-3' sequence is used, and the matched sequence 5'-GTAGCAT-3' sequence is used, so that when there is no aligned and matched sequence except for the first gene and the second gene, the sequence can be selected as a dual target nucleic acid molecule.

In the present invention, the dual target nucleic acid molecule may be designed as siRNA or shRNA, but is not limited thereto. When designed as siRNA, it may be a double-stranded siRNA of 17 to 24 bp, and one strand may be derived from a "segmented sequence", that is, derived from a first gene, and the other strand may be derived from a "matched sequence", that is, derived from a second gene. When designed as shRNA, it may be designed as a structure including a sequence derived from a first gene, a structure capable of forming a hairpin structure, and a sequence derived from a second gene.

In the present invention, targeting the first gene or the second gene may mean targeting the transcript of the first gene or the transcript of the second gene.

The nucleic acid molecules of the present invention are intended to target different genes, and the first gene targeted by the segmented sequence and the second gene targeted by the "matched sequence" may be genes associated with the same disease, for example, cancer, i.e., oncogenes, but are not limited thereto.

FIG. 6 is a block diagram of a device (200) for designing a dual target nucleic acid molecule according to one embodiment.

Referring to FIG. 6, the device (900) may include a memory (210), an input unit (220), and at least one processor (230). According to the method of designing a nucleic acid molecule proposed in the above embodiments, the memory (210), the input unit (220), and at least one processor (230) may operate. However, the components of the device (200) according to one embodiment are not limited to the above-described example. According to another embodiment, the device (200) for designing a nucleic acid molecule may include more or fewer components than the above-described components.

The memory (210) according to one embodiment can store a database that has been previously created based on the sequence information of a gene. For example, the memory (210) can store data of NCBI as an example of a database, and can store information on already formed dual specific nucleic acid molecules. In addition, according to another embodiment, the memory (210) can store genetic information obtained through NCBI.

According to one embodiment, the input unit (220) can input target gene names or sequence information, etc. However, this is only one embodiment, and the input unit (220) can receive all user inputs required for dual target nucleic acid sequence design.

At least one processor (230) according to one embodiment may generate sequence information through a segmentation step of segmenting sequence information from the 5' end of mRNA of the first gene into lengths of 15 to 30 mer using a pre-generated database, and the segmentation step may be a multiple-time segmentation of 15 to 30 mer lengths, starting from a point sequentially spaced apart by 1 nt (nucleotide) in the 3' end direction from the 5' end of the mRNA;

(2) The sequence information can be generated through a segmentation step of segmenting the sequence information from the 3' end of the mRNA of the second gene into segments of 15 to 30 mer in length, and the segmentation step can be segmented multiple times into segments of 15 to 30 mer in length, starting from points sequentially spaced by 1 nt each in the 5' end direction from the 3' end of the mRNA, and the segmented sequences can be aligned to score the matched sequences, and sequences with high scores can be selected according to specific criteria to determine them as candidates for the dual target nucleic acid molecule.

For the above scoring, the scoring method disclosed in the existing Republic of Korea application 10-2019-0610720 can be used.

In addition, when a mismatch exists at a position corresponding to 0.25 to 0.40 or a position corresponding to 0.60 to 0.75 from the 5' end or the 3' end with respect to the total nucleotide (nt) length of 1.00 of the determined candidate substance, an optimal dual target nucleic acid molecule can be derived.

A weight may be assigned when a mismatch exists at a position corresponding to 0.25 to 0.30 or a position corresponding to 0.70 to 0.75 from the 5' end or the 3' end of the sequence matching the segmented sequence.

The device according to the present invention may include a processor, a memory for storing and executing program data, a permanent storage such as a disk drive, a communication port for communicating with an external device, a user interface device such as a touch panel, a key, a button, etc. The methods implemented as software modules or algorithms may be stored on a computer-readable recording medium as computer-readable codes or program commands executable on the processor. Here, the computer-readable recording medium includes a magnetic storage medium (e.g., a read-only memory (ROM), a random-access memory (RAM), a floppy disk, a hard disk, etc.) and an optical reading medium (e.g., a CD-ROM, a Digital Versatile Disc (DVD)). The computer-readable recording medium may be distributed to computer systems connected to a network, so that the computer-readable code may be stored and executed in a distributed manner. The medium is readable by a computer, stored in a memory, and executed by a processor.

All documents, including published documents, patent applications, patents, etc., cited in the present invention may be incorporated into the present invention to the same extent as if each cited document were individually and specifically indicated to be incorporated or collectively indicated to be incorporated into the present invention as a whole.

For the purpose of understanding the present invention, reference numerals have been given to preferred embodiments illustrated in the drawings, and specific terms have been used to describe the embodiments of the present invention; however, the present invention is not limited by the specific terms, and the present invention may include all components that can be commonly conceived by those skilled in the art.

The present invention may be represented by functional block configurations and various processing steps. These functional blocks may be implemented by a variety of hardware and/or software configurations that perform specific functions. For example, the present invention may employ direct circuit configurations such as memory, processing, logic, look-up tables, etc., which may perform various functions under the control of one or more microprocessors or other control devices. Similarly to the fact that the components of the present invention may be implemented by software programming or software components, the present invention may be implemented by programming or scripting languages such as C, C++, Java, assembler, R, Python, etc., including various algorithms implemented by a combination of data structures, processes, routines, or other programming configurations. The functional aspects may be implemented by algorithms that are executed on one or more processors. In addition, the present invention may employ conventional techniques for electronic environment setting, signal processing, and/or data processing. Terms such as "mechanism", "element", "means", and "configuration" may be used broadly and are not limited to mechanical and physical configurations. The terms may also include the meaning of a series of software processes (routines) in connection with a processor, etc.

The specific implementations described in the present invention are only exemplary embodiments and do not limit the scope of the present invention in any way. For the sake of brevity of the specification, descriptions of conventional electronic components, control systems, software, and other functional aspects of the systems may be omitted. In addition, the connections or lack of connections of lines between components illustrated in the drawings are merely exemplary of functional connections and/or physical or circuit connections, and may be replaced or represented as various additional functional connections, physical connections, or circuit connections in an actual device. In addition, if there is no specific mention such as “essential,” “important,” etc., the component may not be absolutely necessary for the application of the present invention.

The use of the term "above" and similar referential terms in the specification of the present invention (especially in the claims) may be in both singular and plural. In addition, when a range is described in the present invention, it is intended to include inventions that apply individual values belonging to the range (unless otherwise stated), and it is the same as stating each individual value constituting the range in the detailed description of the invention. Finally, unless the order of the steps constituting the method according to the present invention is explicitly stated or stated to the contrary, the steps may be performed in any suitable order. The invention is not necessarily limited by the order in which the steps are described. The use of all examples or exemplary terms (e.g., etc.) in the present invention is merely intended to describe the invention in detail, and the scope of the invention is not limited by the examples or exemplary terms unless otherwise defined by the claims. In addition, those skilled in the art will recognize that various modifications, combinations, and alterations may be made according to design conditions and factors within the scope of the appended claims or their equivalents.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to concretize the content of the present invention and the present invention is not limited thereto.

Example 1. Design of dual target siRNA

After extracting the mRNA sequences of two types of target genes (the first gene and the second gene), complementary sequences were created therefrom, and the first gene was segmented into 17 to 24 mer lengths of 1 nt (nucleotide) each in the 3' to 5' direction of the sequence complementary to the mRNA sequence, and the other second gene was segmented into 17 to 24 mer lengths of 1 nt each in the 5' to 3' direction of the sequence complementary to the mRNA sequence. For each of the two segmented gene sequences, alignment was performed with approximately 37,961 gene sequences provided by NCBI using the Striped Smith-Waterman algorithm. After this, if the segmented sequence in the mRNA of the first gene 1) is not located within a distance of 75 nt from the start codon, 2) if the content of G base and C base is 36% to 52% with respect to the total number of bases, 3) if the GC repeat sequence is less than 3, 4) if the AT repeat sequence is less than 4, 5) if the segmented sequence is a sense sequence, if the G base or C base is present at the first position, 6) if the segmented sequence is a sense sequence, if the A base is present at the third position, 7) if the segmented sequence is a sense sequence, if the T or U base is present at the 10th position, 8) if the segmented sequence is a sense sequence, if the G base is not present at the 13th position, 9) if the segmented sequence is a sense sequence, if the A base is present at the 19th position, 10) if the segmented sequence is a sense sequence A weighting of 1 point was given to the following cases: 1) if the segmented sequence is an antisense sequence and there is no A base, T base, or U base at the first position; 12) if the segmented sequence is an antisense sequence and there is a G base at the first position of the sequence; 13) if the segmented sequence is an antisense sequence and there is A at the 6th position; 14) if the segmented sequence is an antisense sequence and the 19th sequence is not a G base or a C base; 15) if the segmented sequence is an antisense sequence and the 19th sequence is a U base or a T base; and 16) if the segmented sequence is located in the coding sequence (CDS); And the aligned and matched sequence is also a sequence in which the segmented sequence in the mRNA of the second gene targeted by the matched sequence is 1) not located within a distance of 75 bp from the start codon, 2) if the content of G base and C base is 36% to 52% with respect to the total number of bases, 3) if the GC repeat sequence is less than 3, 4) if the AT repeat sequence is less than 4, 5) if the segmented sequence is a sense sequence, if the G base or C base is present at the first position, 6) if the segmented sequence is a sense sequence, if the A base is present at the third position, 7) if the segmented sequence is a sense sequence, if the T or U base is present at the 10th position, 8) if the segmented sequence is a sense sequence, if the G base is not present at the 13th position, 9) if the segmented sequence is a sense sequence, if the A base is present at the 19th position. 10) If the segmented sequence is a sense sequence and there is no G base or C base at the 19th position, 11) If the segmented sequence is an antisense sequence and there is no A base, T base, or U base at the first position, 12) If the segmented sequence is an antisense sequence and there is a G base at the first position of the sequence, 13) If the segmented sequence is an antisense sequence and there is A at the 6th position, 14) If the segmented sequence is an antisense sequence and the 19th sequence is not a G base or C base, 15) If the segmented sequence is an antisense sequence and the 19th sequence is a U base or T base, and 16) If the segmented sequence is located in a coding sequence (CDS), a weight of 1 point was given. 99 pairs of dual-specific siRNAs of 17-24 mer length that are complementary to each other while complementarily binding to the mRNA sequences of the first and second genes thus generated were produced (Fig. 1).

Example 2. Design of dual-target siRNAs containing mismatches

Since RISC generally prefers only one of the two siRNA strands, the two sequences must bind to RISC at a similar ratio in order to appropriately down-regulate the mRNAs of two types of genes. However, in the case of conventional dual-target siRNA, the sense sequence, which is a nucleic acid sequence complementary to the mRNA sequences of each gene, i.e., guide RNA (a nucleic acid sequence complementary to the mRNA of the first gene), and the antisense sequence, i.e., passenger RNA (a nucleic acid sequence complementary to the mRNA of the second gene), are 100% complementary in each base, which causes off-target effects. Here, we designed siRNAs to have mismatches at various locations between the guide RNA and passenger RNA sequences in order to control the off-target effect and knock down both types of genes, considering that the number and location of mismatches would be important for RISC to recognize the two strands at a similar ratio (Fig. 2), and then analyzed the correlation between the mismatch locations and the gene expression inhibition effect of the corresponding sequences. Specifically, in 99 pairs of dual target siRNAs (a total of 198 nucleic acid sequences) with lengths ranging from 17 to 24 mer derived for various gene sets in Example 1, the locations of the mismatches between the two sequences were expressed as 'locations relative to the total sequence length (mismatch location)' to standardize their different lengths (Fig. 3a), and the mismatch locations and inhibition efficiencies ( _KDeff ) of a total of 198 sequences were datafied (Fig. 3b). At this time, the mismatch location was divided into intervals of 0.05 (eg 0.00~0.05, 0.05~0.10, …0.90~0.95, 0.95~1.00), and in order to analyze the difference in suppression efficiency according to the presence or absence of mismatch in each interval, the difference in suppression efficiency between the top 30% and bottom 30% of the suppression efficiency of the sequence with mismatch and the top 30% and bottom 30% of the suppression efficiency of the sequence without mismatch was calculated using mathematical equations 1 and 2. In the following mathematical expressions 1 and 2, if there is no effect of mismatch, the results of the upper 30% and the lower 30% are 1, and if there is a difference, 1 is not output, so the difference in the results of the two mathematical expressions above (the relative KD _eff difference according to the mismatch location) was analyzed using the following mathematical expression 3 (a positive value if the mismatch enhances the expression inhibition efficiency of two genes/a negative value if the mismatch decreases the expression inhibition efficiency of two genes), and the effect of the mismatch location of 99 pairs of siRNAs (a total of 198 sequences) on the gene expression inhibition efficiency of bispecific siRNA was analyzed.

As a result, based on the single-stranded sequence of the siRNA pair, when the total length of the siRNA is assumed to be 1.0, when mismatches exist at nucleotides at positions 0.25 to 0.30 and 0.70 to 0.75 from the 5' end (for example, in the case of 20mer siRNA, a mismatch exists at the 14th nucleotide position from the 5' end), the gene silencing efficiency was found to increase (Fig. 4). In addition, it was found that an asymmetric mismatch structure increases the selectivity of siRNA efficacy, and a single-stranded siRNA with a mismatch at position 0.3 tended to be selected as the working sequence, and the other strand with a complementary mismatch at position 0.7 tended to be selected (Fig. 5). As a result, it was confirmed that position 0.3 is the most important site for siRNA efficacy.

Claims (19)

1) a first nucleic acid molecule targeting the mRNA of the first gene; and
2) In a dual target nucleic acid molecule comprising a second nucleic acid molecule targeting the mRNA of a second gene,
The first nucleic acid molecule and the second nucleic acid molecule are partially complementary, and
A dual target nucleic acid molecule, characterized in that base pairs of the first nucleic acid molecule and the second nucleic acid molecule are mismatched at a position corresponding to 0.25 to 0.40, or a position corresponding to 0.60 to 0.75 from the 5' end or the 3' end of the first nucleic acid molecule or the second nucleic acid molecule with respect to a total nucleotide (nt) length of 1.00 of the first nucleic acid molecule or the second nucleic acid molecule. A dual target nucleic acid molecule in claim 1, wherein the first nucleic acid molecule has a sequence that is 100% complementary to the mRNA of the first gene, and the second nucleic acid molecule has a sequence that is 100% complementary to the mRNA of the second gene. In the first paragraph, a dual target nucleic acid molecule, wherein the first nucleic acid molecule and the second nucleic acid molecule have 1 to 3 base pairs of mismatch. A dual target nucleic acid molecule in claim 1, wherein the first nucleic acid molecule or the second nucleic acid molecule is 14 to 30 mer. A dual target nucleic acid molecule in claim 4, wherein the first nucleic acid molecule or the second nucleic acid molecule is 17 to 24 mer. A dual target nucleic acid molecule in claim 1, wherein the dual target nucleic acid molecule is siRNA or shRNA. In the first paragraph, if the dual target nucleic acid molecule is 20 mer,
1) The base at the 7th position from the 5' end of the first nucleic acid molecule and the base at the 14th position from the 5' end of the second nucleic acid molecule are mismatched;
2) the base at the 9th position from the 5' end of the first nucleic acid molecule and the base at the 12th position from the 5' end of the second nucleic acid molecule are mismatched; and
3) A dual target nucleic acid molecule in which the base at the 14th position from the 5' end of the first nucleic acid molecule and the base at the 7th position from the 5' end of the second nucleic acid molecule are mismatched. In a database of malformed dual target nucleic acid molecules,
A method for designing a dual target nucleic acid molecule, comprising the step of selecting a base pair mismatch between a first nucleic acid molecule and a second nucleic acid molecule at a position corresponding to 0.25 to 0.40 or a position corresponding to 0.60 to 0.75 from the 5' or 3' end of the first nucleic acid molecule or the second nucleic acid molecule with respect to a total nucleotide (nt) length of 1.00 of the first nucleic acid molecule or the second nucleic acid molecule of the dual target nucleic acid molecule. A method for designing a dual target nucleic acid molecule in claim 8, wherein the first nucleic acid molecule has a sequence that is 100% complementary to the mRNA of the first gene, and the second nucleic acid molecule has a sequence that is 100% complementary to the mRNA of the second gene. A method for designing a dual target nucleic acid molecule, wherein the first nucleic acid molecule and the second nucleic acid molecule have 1 to 3 base pairs of mismatch in the 8th paragraph. A method for designing a dual target nucleic acid molecule, wherein the first nucleic acid molecule or the second nucleic acid molecule is 14 to 30 mer in the 8th paragraph. A method for designing a dual target nucleic acid molecule, wherein the first nucleic acid molecule or the second nucleic acid molecule is 17 to 24 mer in the 11th paragraph. A method for designing a dual target nucleic acid molecule, wherein the dual target nucleic acid molecule in claim 8 is siRNA or shRNA. In the 8th paragraph, if the dual target nucleic acid molecule is 20mer,
1) The base at the 7th position from the 5' end of the first nucleic acid molecule and the base at the 14th position from the 5' end of the second nucleic acid molecule are mismatched;
2) the base at the 9th position from the 5' end of the first nucleic acid molecule and the base at the 12th position from the 5' end of the second nucleic acid molecule are mismatched; and
3) A method for designing a dual target nucleic acid molecule in which the base at the 14th position from the 5' end of a first nucleic acid molecule and the base at the 7th position from the 5' end of a second nucleic acid molecule are mismatched. In the 8th paragraph, the creation of the dual target nucleic acid molecule database comprises:
A segmentation step is performed to generate sequence information by segmenting the sequence information from the 5' end of the mRNA of the first gene into 15 to 30mer lengths.
The above segmentation step is,
A method for designing a dual target nucleic acid molecule, characterized in that sequence information is generated through multiple segmentation steps of 15 to 30 mer in length, starting from points sequentially spaced by 1 nt (nucleotide) in the 3' direction from the 5' end of the mRNA. In the 8th paragraph, the creation of the dual target nucleic acid molecule database comprises:
A segmentation step is performed to generate sequence information by segmenting the sequence information from the 3' end of the mRNA of the second gene into 15 to 30mer lengths.
The above segmentation step is,
A method for designing a dual target nucleic acid molecule, characterized in that sequence information is generated through multiple segmentation steps of 15 to 30 mer in length, starting from points sequentially spaced by 1 nt in the 5' direction from the 3' end of the mRNA. A method for designing a dual target nucleic acid molecule, wherein the sequence information of the segmentation step of claim 15 or 16 is generated to have a length of 17 to 21 mer. In the 8th paragraph, the creation of the dual target nucleic acid molecule database comprises:
(1) A segmentation step is performed to generate sequence information by segmenting the sequence information from the 5' end of the mRNA of the first gene into segments of 15 to 30 mer in length.
The above segmentation step is,
Sequence information is generated through multiple segmentation steps of 15 to 30 mer length, starting from points sequentially spaced by 1 nt (nucleotide) in the 3' direction from the 5' end of the mRNA;
(2) A segmentation step is performed to generate sequence information by segmenting the sequence information from the 3' end of the mRNA of the second gene into 15 to 30mer lengths.
The above segmentation step is,
Sequence information is generated through multiple segmentation steps of 15 to 30 mer length, starting from a point sequentially spaced by 1 nt in the 5' direction from the 3' end of the mRNA; and
(3) A method for designing a dual target nucleic acid molecule, comprising a step of aligning sequence information generated from mRNA of the first gene and sequence information generated from mRNA of the second gene. A method for designing a dual target nucleic acid molecule, further comprising a step of scoring matched sequences after the aligning step in claim 18.