KR-20260062111-A - Probes for detecting single nucleotide polymorphisms in regions with clustered variants and methods for using thereof

Abstract

The present invention relates to a probe for detecting single nucleotide polymorphisms in a region where mutations are accumulated, and a method for using the same. The system according to the present invention can successfully distinguish single nucleotide polymorphisms (SNPs) by positioning mutations at the 3' end of a reporter probe in batches without a complex design process, and can effectively detect the mutation to be distinguished even if mutations are accumulated in the target area, as there is almost no influence from surrounding mismatches. In addition, since the SNP diagnostic results can be verified on the LFA by applying the system according to the present invention to the LFA, there is an advantage of effectively detecting target SNPs in areas where mutations are accumulated in the field using actual clinical samples.

Inventors

• 이정욱
• 우창하
• 이정민
• 성도언
• 김보경

Assignees

• 포항공과대학교 산학협력단

Dates

Publication Date

20260507

Application Date

20241025

Claims (19)

1. A composition for detecting a target single nucleotide polymorphism (SNP), comprising: a set of SNIPR probes for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated, including a first probe and a second probe; The first probe above is a promoter probe (PP) having the structure of the following general formula I; 3'-X- Y-5' (I) In the above general formula (I), The above X is a stem-loop structure region containing a promoter sequence recognizable by RNA polymerase; the above Y is an Upstream Hybridization Sequence (UHS) region having a hybridization sequence complementary to the target nucleic acid sequence; the above target nucleic acid sequence is DNA or RNA; and the above X and Y are deoxyribonucleotides; The second probe above is a reporter probe (RP) having the structure of the following general formula II; 3'-Y'-Z-5' (II) In the above general formula (II), The above Y' is a Downstream Hybridization Sequence (DHS) region having a hybridization sequence complementary to the target nucleic acid sequence; the above Z is a sequence region having an interactive labeling system comprising one or more labels that generate a detectable signal; the target nucleic acid sequence is DNA or RNA; and the above Y' and Z are deoxyribonucleotides; The above UHS and DHS have hybridization sequences complementary to the target nucleic acid sequence having a length of 16 nt to 150 nt; The UHS (Upstream Hybridization Sequence) region of the first probe or the DHS (Downstream Hybridization Sequence) region of the second probe is designed such that there is no mismatch in the nucleotide corresponding to the -1 or +1 position when the position of the target single nucleotide polymorphism (SNP) is set to 0, A composition further comprising all probe combinations designed such that adenine, cytosine, guanine, and thymine are located at the -1 or +1 positions, respectively.
2. A composition for detecting a target single nucleotide polymorphism (SNP), comprising: an isothermal one-pot reaction SNIPR probe set for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated, including a first probe and a second probe; and a nucleic acid for interfering with double-strand recombination of at least one target nucleic acid sequence. The first probe above is a promoter probe (PP) having the structure of the following general formula I; 3'-X- Y-5' (I) In the above general formula (I), The above X is a stem-loop structure region containing a promoter sequence recognizable by RNA polymerase; the above Y is an Upstream Hybridization Sequence (UHS) region having a hybridization sequence complementary to the target nucleic acid sequence; the above target nucleic acid sequence is DNA or RNA; and the above X and Y are deoxyribonucleotides; The second probe above is a reporter probe (RP) having the structure of the following general formula II; 3'-Y'-Z-5' (II) In the above general formula (II), The above Y' is a Downstream Hybridization Sequence (DHS) region having a hybridization sequence complementary to the target nucleic acid sequence; the above Z is a sequence region having an interactive labeling system comprising one or more labels that generate a detectable signal; the target nucleic acid sequence is DNA or RNA; and the above Y' and Z are deoxyribonucleotides; The above UHS and DHS have hybridization sequences complementary to the target nucleic acid sequence having a length of 16 nt to 150 nt; The nucleic acid for interfering with the double-strand rejunction of the above target nucleic acid sequence is a sequence complementary to the target nucleic acid sequence at the 3' end or 5' end position of the complementary binding region of the SNIPR probe set; The UHS (Upstream Hybridization Sequence) region of the first probe or the DHS (Downstream Hybridization Sequence) region of the second probe is designed such that there is no mismatch in the nucleotide corresponding to the -1 or +1 position when the position of the target single nucleotide polymorphism (SNP) is set to 0, A composition further comprising all probe combinations designed such that adenine, cytosine, guanine, and thymine are located at the -1 or +1 positions, respectively.
3. A composition for detecting a target single nucleotide polymorphism (SNP), comprising: a set of SNIPR probes for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated, comprising a first probe and a second probe; and a detection probe comprising a third probe and a fourth probe. The first probe above is a promoter probe (PP) having the structure of the following general formula I; 3'-X- Y-5' (I) In the above general formula (I), The above X is a stem-loop structure region containing a promoter sequence recognizable by RNA polymerase; the above Y is an Upstream Hybridization Sequence (UHS) region having a hybridization sequence complementary to the target nucleic acid sequence; the above target nucleic acid sequence is DNA or RNA; and the above X and Y are deoxyribonucleotides; The second probe above is a reporter probe (RP) having the structure of the following general formula II; 3'-Y'-Z-5' (II) In the above general formula (II), The above Y' is a DHS (Downstream Hybridization Sequence) site having a hybridization sequence complementary to the target nucleic acid sequence; the above Z is a site having an arbitrary sequence for imparting an arbitrary sequence to the transcript; the above target nucleic acid sequence is DNA or RNA; and the above Y' and Z are deoxyribonucleotides; The above UHS and DHS have hybridization sequences complementary to the target nucleic acid sequence having a length of 16 nt to 150 nt; The third probe has a sequence complementary to the transcript transcribed by the first probe and the second probe, and has a structure in which an anti-ligand is bound to the 3'end; it is a detection probe (DP) that binds to the transcript transcribed by the first probe and the second probe; The fourth probe has a sequence complementary to the transcript transcribed by the first probe and the second probe, and has a structure in which an anti-ligand target substance is bound to the 5' end, and is a detection probe (DP) that binds to the transcript transcribed by the first probe and the second probe; The UHS (Upstream Hybridization Sequence) region of the first probe or the DHS (Downstream Hybridization Sequence) region of the second probe is designed such that there is no mismatch in the nucleotide corresponding to the -1 or +1 position when the position of the target single nucleotide polymorphism (SNP) is set to 0, A composition further comprising all probe combinations designed such that adenine, cytosine, guanine, and thymine are located at the -1 or +1 positions, respectively.
4. A composition for detecting a target single nucleotide polymorphism (SNP), comprising: a set of isothermal one-pot reaction SNIPR probes for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated, comprising a first probe and a second probe; a detection probe comprising a third probe and a fourth probe; and at least one nucleic acid for interfering with double-strand recombination of a target nucleic acid sequence. The first probe above is a promoter probe (PP) having the structure of the following general formula I; 3'-X- Y-5' (I) In the above general formula (I), The above X is a stem-loop structure region containing a promoter sequence recognizable by RNA polymerase; the above Y is an Upstream Hybridization Sequence (UHS) region having a hybridization sequence complementary to the target nucleic acid sequence; the above target nucleic acid sequence is DNA or RNA; and the above X and Y are deoxyribonucleotides; The second probe above is a reporter probe (RP) having the structure of the following general formula II; 3'-Y'-Z-5' (II) In the above general formula (II), The above Y' is a DHS (Downstream Hybridization Sequence) site having a hybridization sequence complementary to the target nucleic acid sequence; the above Z is a site having an arbitrary sequence for imparting an arbitrary sequence to the transcript; the above target nucleic acid sequence is DNA or RNA; and the above Y' and Z are deoxyribonucleotides; The above UHS and DHS have hybridization sequences complementary to the target nucleic acid sequence having a length of 16 nt to 150 nt; The third probe has a sequence complementary to the transcript transcribed by the first probe and the second probe, and has a structure in which an anti-ligand is bound to the 3'end; it is a detection probe (DP) that binds to the transcript transcribed by the first probe and the second probe; The fourth probe has a sequence complementary to the transcript transcribed by the first probe and the second probe, and has a structure in which an anti-ligand target substance is bound to the 5' end, and is a detection probe (DP) that binds to the transcript transcribed by the first probe and the second probe; The nucleic acid for interfering with the double-strand rejunction of the above target nucleic acid sequence is a sequence complementary to the target nucleic acid sequence at the 3' end or 5' end position of the complementary binding region of the SNIPR probe set; The UHS (Upstream Hybridization Sequence) region of the first probe or the DHS (Downstream Hybridization Sequence) region of the second probe is designed such that there is no mismatch in the nucleotide corresponding to the -1 or +1 position when the position of the target single nucleotide polymorphism (SNP) is set to 0, A composition further comprising all probe combinations designed such that adenine, cytosine, guanine, and thymine are located at the -1 or +1 positions, respectively.
5. A composition according to claim 1 or 2, wherein the label of the second probe is any one selected from the group consisting of a chemical label, an enzyme label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, and a metal label.
6. A composition according to any one of claims 1 to 4, wherein the variation is selected from any one of viruses, harmful bacterial strains, and genetic variations of animals including humans.
7. A composition according to claim 1, wherein the Z region of the second probe is designed so as not to undergo a cross-coupling reaction with the first probe.
8. A composition according to claim 2, wherein the Z region of the second probe is designed so as not to undergo a cross-coupling reaction with the nucleic acid for interfering with the double-strand recombination of the first probe and the target nucleic acid sequence.
9. A composition according to paragraph 3, wherein the Z region of the second probe is designed so as not to undergo a cross-coupling reaction with the first probe, the third probe, and the fourth probe.
10. A composition according to claim 4, wherein the Z region of the second probe is designed so as not to undergo a cross-coupling reaction with the first probe, the third probe, the fourth probe, and the nucleic acid for interfering with the double-strand rejoining of the target nucleic acid sequence.
11. A composition according to claim 3 or 4, wherein the third probe and the fourth probe are linear and designed so as not to cause a cross-coupling reaction between the two probes, the SNIPR probe set, and the nucleic acid for interfering with the double-strand recombination of the target nucleic acid sequence.
12. A composition according to claim 3 or 4, wherein the anti-ligand target substance of the fourth probe is any one selected from the group consisting of ALEX-350, FAM, VIC, TET, CAL Fluor®540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, and Quasar 705, a fluorescent probe, a substrate, a protein capable of specific detection of an anti-ligand, or a nanoparticle capable of specific detection of an anti-ligand.
13. A composition according to claim 2 or 4, wherein the nucleic acid for interfering with the double-strand rejoining of a target nucleic acid sequence is one or two.
14. A kit for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated, comprising: a composition according to any one of claims 1 to 13; a ligation agent; and an RNA polymerase.
15. In claim 14, the kit further comprises an isothermal single reaction solution; or a lateral flow assay (LFA) strip.
16. In claim 15, the above-mentioned isothermal single-reaction solution is any one selected from the group consisting of Tris-HCl, MgCl2, NTPs, NaCl, and ET-SSB (Extreme Thermostable Single-Stranded DNA Binding Protein), a kit.
17. (a) a step of adding a composition according to any one of claims 1 to 13 to a target nucleic acid sequence; and (b) a step of detecting signal generation; a method for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated.
18. (i) a step of separating the double strand of the target nucleic acid sequence at a high temperature of 80~100℃; (ii) a step of cooling the reactant of (a) to 20°C or lower, adding a composition according to claim 1 or 2, and carrying out an isothermal single reaction at 15 to 50°C; and (iii) a step of detecting signal generation; a method for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated.
19. (a) A step of adding the composition according to paragraph 3 or 4 to a target nucleic acid sequence, and then transcribing to obtain a reaction product; (b) loading the reaction product according to step (a) above onto one side of a lateral flow analysis device to form a complex of metal nanoparticles conjugated with an antibody that specifically binds to the anti-ligand of the third probe and the anti-ligand target substance of the fourth probe; and (c) a step of confirming whether a band is formed by the formation of a complex of metal nanoparticles conjugated with an antibody that specifically binds to the anti-ligand target substance of the fourth probe of step (b); comprising a method for detecting a target single nucleotide polymorphism (SNP) in a region where mutations are accumulated.

Description

Probes for detecting single nucleotide polymorphisms in regions with clustered variants and methods for using thereof The present invention relates to a probe for detecting single nucleotide polymorphisms in a region where mutations are accumulated, and a method for using the same. Rapid and accurate disease diagnosis is the most critical factor in patient treatment. While previous diagnostic methods relied on patient symptoms and physician experience, recent approaches are becoming more objective and precise. In particular, the field of molecular diagnostics is recognized as having shifted the paradigm of medical technology, as it enables the specific and objective detection and monitoring of diseases by detecting changes at the molecular level, such as in nucleic acids. Meanwhile, the recent COVID-19 outbreak has demonstrated the power of molecular diagnostic technology while simultaneously revealing its limitations. Although molecular diagnostics allowed for the accurate identification of infected individuals to enable measures such as treatment or isolation, low throughput and slow diagnostic speeds became problematic in situations where a large number of patients converge, such as during a pandemic. This issue became particularly evident following the emergence of highly contagious variants like Delta and Omicron; current molecular diagnostic technology was unable to rapidly identify these variants on-site, failing to prevent their spread early or efficiently handle the surge in patients caused by these variants. Furthermore, as it has been shown that some variants reduce the effectiveness of antibodies or treatments already formed from previous infections or vaccines, technologies capable of detecting and monitoring these variants have garnered significant attention in the field of treatment. Current SNP classification methods are based on sequencing, specifically whole genome sequencing (WGS) technologies such as next-generation sequencing (NGS). However, since diagnosis often requires only a portion of genetic information, WGS can be an inefficient technology in terms of experimental cost and time. In particular, it has the disadvantage of being unsuitable for field application and forcing a centralized diagnostic system, as it requires specialized personnel to be constantly on-site for analysis and specialized equipment. Many technologies have been researched to detect targeted SNPs, and some have even been commercialized. However, PCR-based SNP analysis fundamentally requires a fluorescence reader, and in this case, the high cost of probes is cited as a drawback. Additionally, there is a problem where performance drops significantly if a mismatch is present in the sequence surrounding the SNP to be distinguished. This implies that diagnostic performance is limited when mutations occur in pathogen genes, which can be problematic, particularly when detecting RNA viruses that mutate frequently. The field currently being most actively researched is CRISPR-based technology. In fact, technologies such as SHERLOCK and DETECTR have been developed and utilized for SARS-CoV-2 diagnosis, and it has been revealed that SNP differentiation using CRISPR is possible to some extent in some technologies. However, it is known that the effectiveness of SNP differentiation in CRISPR systems is limited to certain regions of the target sequence. Although CRISPR has some degree of mismatch tolerance, this acts as a weakness in single SNP diagnosis, so it is often necessary to introduce artificial mismatches into crRNA to clearly distinguish signals. Meanwhile, ligase-based SNP detection methods have traditionally been used for SNP differentiation. The reaction rate of DNA ligase varies significantly depending on whether the ligation junction site is complementary, enabling specific SNP differentiation. To date, ligase-based mutation detection methods such as LCR and LDR have been developed. These technologies are generally suitable for SNP differentiation due to their minimal sequence limitations and offer the advantage of high sensitivity (ratio of SNP samples to total samples). However, LCR requires equipment such as thermocyclers, while LDR suffers from the drawback of low sensitivity. The disadvantage of requiring an additional reporting process separate from the reaction has also limited the potential for using ligase-based detection methods in point-of-care diagnostics. Accordingly, the inventors completed the present invention by developing a technology (SplintR-ligase based SNP discrimination reaction; SNIPR) that can quickly and simply detect SNPs in dsDNA in the field using an existing SENSR system. Figure 1 is a schematic diagram showing the configuration of a Field-deployable ligation-dependent SNP discrimination reaction (SNIPR) system according to the present invention. FIG. 2 is a schematic diagram showing a detection method that can be utilized by applying a Field-deployable ligation-dependent SNP discrim