CN116024192A - A TbAgo-based nucleic acid cutting system and detection method and kit for target nucleic acid molecules - Google Patents

Abstract

The invention provides a TbAgo-based nucleic acid cleavage system, a target nucleic acid molecule detection method and a kit, wherein the target nucleic acid molecule detection method comprises the steps of adding Ago protein, guide DNA and a nucleic acid probe into a reaction system containing target nucleic acid molecules to be detected, and detecting the target nucleic acid molecules after the reaction is completed. The method can be used for rapidly detecting pathogenic microorganisms, gene mutation, specific target DNA and the like.

Description

A nucleic acid cleavage system based on TbAgo and a detection method and kit for target nucleic acid molecules

Technical Field

The present invention belongs to the field of biotechnology, and specifically relates to a nucleic acid cleavage system based on a novel medium- and high-temperature Argonaute protein TbAgo, and a detection method and kit for target nucleic acid molecules developed thereby.

Background Art

Nucleic acid detection technology has been widely used in various fields, such as biomolecular medical diagnosis, food safety testing, environmental monitoring, pathogenic microorganism detection, genotyping, etc.

Although traditional nucleic acid detection technologies, such as quantitative polymerase chain reaction (qPCR), high-throughput sequencing, and southern blot, are still widely used, these technologies have their own drawbacks, such as being expensive, time-consuming, and requiring professional personnel to operate.

In recent years, the CRISPR/Cas system, i.e., Clustered regularly interspaced short palindromic repeats/CRISPR associated gene (CRISPR/Cas), has become a hot topic of research, and nucleic acid detection technologies based on this system have also been continuously developed, such as SHERLOCK, DETECTOR, HOLMES, etc. Although more economical and convenient than traditional nucleic acid detection technologies, they still have certain defects. For example, the recognition and cutting of target nucleic acid molecules by Cas proteins rely on PAM sites, which limits the sites they can act on; Cas proteins can only function under the mediation of guide RNA (gRNA), and RNA is not only complicated in production process and expensive, but also easily degraded by nucleases in the environment, making the stability of the entire system poor; the trans-cutting activity stimulated by Cas proteins after recognizing and binding to specific nucleic acids is non-specific nuclease activity, so the nucleic acid detection technology developed based on this cannot meet the requirements of detecting multiple target nucleic acid molecules with a single system.

In summary, it is still necessary to develop a new nucleic acid detection technology that has no restrictions on detection sites, a stable system, and can detect multiple target nucleic acid molecules at the same time.

Argonaute (Ago) protein was first mentioned in a study describing Arabidopsis mutants. Currently reported Ago proteins are mainly divided into two categories: eukaryotic Ago (eAgo) and prokaryotic Ago (pAgo). eAgo is a key player in the RNA interference (RNAi) pathway in eukaryotes, which can regulate gene expression after transcription, thereby defending against invading RNA viruses and protecting the integrity of the genome. pAgo can bind to single-stranded guide DNA (gDNA) or guide RNA (gRNA) and cut DNA or RNA complementary to the guide nucleic acid sequence at specific sites, that is, cutting between the 10th and 11th positions from the 5' end of the guide nucleic acid, which is believed to be an immune mechanism of prokaryotes.

Currently, only a very small number of pAgos have been characterized. Most of these Ago proteins are high-temperature Agos, which can bind to single-stranded gDNA or gRNA at high temperatures and cut single-stranded DNA or single-stranded RNA. Compared with the CRISPR/Cas system, pAgo does not rely on the PAM site when cutting the target nucleic acid molecule chain. Therefore, the purpose of cutting nucleic acids of any sequence can be achieved through the design of gDNA; and gDNA has better stability than gRNA; at the same time, because the cutting behavior depends on the specific sequence of gDNA, multiple cutting reactions in a single reaction system do not interfere with each other, and the purpose of simultaneously detecting multiple target nucleic acid molecules in a single system can be achieved.

Based on the shortcomings of existing nucleic acid detection technologies and the characteristics of the pAgo system, the development of new nucleic acid detection technologies based on pAgo proteins has important practical significance and broad application prospects.

Summary of the invention

In a first aspect of the present invention, a nucleic acid cleavage system is provided.

The nucleic acid cleavage system comprises:

(a) Programmable endonuclease Argonaute (Ago); the programmable endonuclease Argonaute is derived from Thermus brockianus, and the programmable endonuclease Argonaute is the programmable endonuclease TbAgo.

(b) guide DNA (gDNA); and

(c) a nucleic acid probe, wherein if the nucleic acid probe is cleaved, the cleavage is detectable.

In another preferred embodiment, the reaction temperature of the nucleic acid cleavage system is 50°C-95°C, preferably 50°C-85°C, and more preferably 50°C-75°C.

In another preferred embodiment, the TbAgo includes wild-type and mutant TbAgo, and the TbAgo has at least 80% identity with the amino acid sequence shown in SEQ ID NO:1.

In another preferred example, the amino acid sequence of the wild-type TbAgo is shown in NCBI sequence number WP_071678161.1.

In another preferred embodiment, the gDNA is a single-stranded DNA molecule with 5'-end phosphorylation or 5'-end hydroxylation.

In another preferred embodiment, the gDNA is a single-stranded DNA molecule with 5' end phosphorylated.

In another preferred embodiment, the gDNA and the nucleic acid probe have reverse complementary fragments, which can guide TbAgo to cut the nucleic acid probe and generate a detectable signal value.

In another preferred embodiment, the length of the gDNA is 10nt-35nt, more preferably 14nt-35nt.

In another preferred embodiment, the nucleic acid probe is single-stranded DNA (ssDNA).

In another preferred embodiment, the nucleic acid probe is unmodified single-stranded DNA.

In another preferred embodiment, when the nucleic acid probe is cleaved, the cleavage can be detected by electrophoresis.

In another preferred embodiment, the electrophoresis method is a 14% nucleic acid Urea-PAG electrophoresis detection method.

In another preferred embodiment, the nucleic acid probe is a single-stranded DNA molecule with a fluorescent group modified only at the 5' end, and the fluorescent group is FAM.

In another preferred embodiment, the nucleic acid probe is a nucleic acid probe having both a fluorescent group and a quenching group.

In another preferred embodiment, the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof.

In another preferred embodiment, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.

In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5' end and the 3' end of the nucleic acid probe.

In another preferred embodiment, when the nucleic acid probe is cleaved, the cleavage can be detected by an enzyme reader.

In another preferred embodiment, the nucleic acid probe is a nucleic acid probe carrying both a fluorescent group and a biotin group, and the fluorescent group is FAM.

In another preferred embodiment, the fluorescent group and the biotin group are independently located at the 5' end and the 3' end of the nucleic acid probe.

In another preferred embodiment, when the nucleic acid probe is cleaved, the cleavage can be detected by a colloidal gold test strip.

In another preferred embodiment, the nucleic acid cleavage system further comprises: (d) divalent metal ions.

In another preferred embodiment, the divalent metal ion is Mn ²⁺ , Mg ²⁺ , Ca ²⁺ , Co ²⁺ , Mn ²⁺ , Zn ²⁺ , Fe ²⁺ or Cu ²⁺ , preferably Mn ²⁺ , Mg ²⁺ or Co ²⁺ , and most preferably Mn ²⁺ .

In another preferred embodiment, in the nucleic acid cleavage system, the concentration of Mn ²⁺ is 0 μM-1000 μM, preferably 100 μM-1000 μM, and more preferably 250 μM-1000 μM.

In another preferred embodiment, the nucleic acid cleavage system further comprises: (e) a buffer.

In another preferred embodiment, the concentration of NaCl in the buffer is 0mM-1000mM, preferably 0mM-500mM, and most preferably 100mM-250mM.

In another preferred example, when in the reverse complementary region between the gDNA and the nucleic acid probe, there is a mismatch with the nucleic acid probe at any base from the 7th to the 14th base from the 5' end and at the 1st, 3rd, and 5th bases, the cleavage rate of the programmable nuclease TbAgo will be significantly reduced.

In another preferred example, the significantly reduced cleavage rate of the programmable endonuclease TbAgo means that under the same reaction conditions, the cleavage rate of the programmable endonuclease TbAgo is reduced by ≥70%, preferably by ≥80%, and more preferably by ≥90%.

In another preferred embodiment, in the nucleic acid cleavage system, the concentration of the fluorescent nucleic acid probe is 0.1 μM-2 μM, preferably 0.5 μM-2 μM, more preferably 0.5 μM-1 μM, and most preferably 0.5 μM.

In another preferred embodiment, in the nucleic acid cleavage system, the concentration of the programmable endonuclease TbAgo is 10 nM-10 μM, preferably 500 nM-5 μM, and most preferably 2.5 μM.

In another preferred embodiment, in the nucleic acid cleavage system, the concentration of the gDNA is 10 nM-10 μM, preferably 100 nM-1 μM, and most preferably 500 nM.

In a second aspect of the present invention, a detection system for a target nucleic acid molecule is provided.

The detection system comprises the nucleic acid cleavage system as described in the first aspect of the present invention.

The target nucleic acid molecule is a DNA molecule or an RNA molecule.

In another preferred embodiment, the detection system further comprises a detection object, namely a target nucleic acid molecule.

In another preferred embodiment, the target nucleic acid molecule in the detection system is derived from pathogenic microorganisms, gene mutations, plants, animals, viruses, and any combination thereof.

In another preferred embodiment, the number of target nucleic acid molecules that can be detected by the detection system is N, and N is a natural number such as 1, 2, 3, 4, 5, etc.

In another preferred embodiment, the target nucleic acid molecule in the detection system includes a synthetic or natural nucleic acid molecule.

In another preferred embodiment, the target nucleic acid molecule in the detection system includes a wild-type or mutant nucleic acid molecule.

In another preferred embodiment, the detection system further comprises a reagent for amplifying the target nucleic acid molecule.

In another preferred embodiment, the guide DNA in the detection system comprises one or more different gDNAs.

In another preferred embodiment, the nucleic acid probe in the detection system comprises one or more different nucleic acid probes.

In another preferred embodiment, the nucleic acid probes in the detection system correspond one-to-one to the target nucleic acid molecules, specifically, they are sequence complementary.

In another preferred embodiment, in the detection system, the programmable endonuclease TbAgo will recognize and cut the target nucleic acid molecule under the guidance of gDNA, thereby generating new gDNA, and the new gDNA is sequence complementary to the corresponding nucleic acid probe.

In another preferred embodiment, in the detection system, the programmable endonuclease TbAgo will recognize and cut the corresponding nucleic acid probe under the guidance of the new gDNA, thereby generating a corresponding detectable signal.

In another preferred example, in the detection system, the concentration of the programmable nuclease TbAgo is 10nM-10μM, preferably 500nM-5μM, and optimally 5μM.

In another preferred embodiment, in the detection system, the concentration of the target nucleic acid molecule is 1fM-200 pM, preferably 1fM-1000fM, and most preferably 1fM-100fM.

In another preferred embodiment, in the detection system, the concentration of the gDNA is 10nM-10μM, preferably 100nM-1μM, and most preferably 250nM.

In another preferred embodiment, in the detection system, the concentration of the nucleic acid probe is 100nM-1000nM, preferably 500nM-1000nM.

In another preferred embodiment, the detection system further comprises (d) divalent metal ions, as described in the first aspect of the present invention.

In another preferred embodiment, the detection system further comprises (e) a buffer, as described in the first aspect of the present invention.

In another preferred embodiment, in the detection system, the working temperature of the programmable nuclease TbAgo is as described in the first aspect of the present invention.

In a third aspect of the present invention, a method for detecting a target nucleic acid molecule for non-diagnostic purposes is provided, comprising the steps of:

(a) providing a sample to be tested;

(b) providing a reaction system to process the sample to be tested and form a reaction solution; the reaction system is a target nucleic acid molecule detection system as described in the second aspect of the present invention;

(c) Detecting the signal of the reaction solution.

Among them, if a specific signal is detected, it means that the test sample contains a specific target nucleic acid molecule; if no specific signal is detected, it means that the test sample does not contain a specific target nucleic acid molecule.

In another preferred embodiment, the reaction system comprises reagents for amplifying target nucleic acid molecules.

In another preferred embodiment, the nucleic acid amplification method is selected from PCR amplification, RT-PCR amplification, LAMP amplification, RT-LAMP amplification, RPA amplification, RT-RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification and rolling circle amplification.

In another preferred embodiment, the method for detecting a target nucleic acid molecule detects a SNP, a point mutation, a deletion or an insertion.

In another preferred embodiment, in step (c), a microplate reader is used to read the signal.

In another preferred embodiment, a colloidal gold test strip is used to read the signal in step (c).

In another preferred embodiment, the method for detecting target nucleic acid molecules is used for in vitro detection.

In another preferred embodiment, the method for detecting target nucleic acid molecules is non-diagnostic and non-therapeutic.

In the fourth aspect of the present invention, a use of an Ago protein in a method for detecting a target nucleic acid molecule is provided, wherein the Ago protein is TbAgo or a similar protein having at least 80% identity with the amino acid sequence shown in SEQ ID NO:1.

In another preferred embodiment, the programmable endonuclease TbAgo includes TbAgo derived from thermophilic bacteria Thermus brockianus; or a homologous protein having similar cleavage activity.

In another preferred example, the programmable nuclease TbAgo includes wild-type and mutant TbAgo.

In another preferred embodiment, the programmable endonuclease TbAgo has an amino acid sequence selected from the following group:

(i) the amino acid sequence shown in NCBI sequence number WP_071678161.1; and

(ii) Based on the sequence shown in NCBI sequence number WP_071678161.1, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 10 amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues) are added to its N-terminus or C-terminus to obtain an amino acid sequence; and the obtained amino acid sequence has ≥80% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98% or ≥99%) sequence identity with the sequence shown in NCBI sequence number WP_071678161.1; and the obtained amino acid sequence has the same or similar function as (i).

In a fifth aspect of the present invention, a detection kit for a target nucleic acid molecule is provided, comprising an Ago protein, a guide DNA, and a nucleic acid probe.

The present invention constructed a recombinant plasmid pET28a-TbAgo by mining and aligning the gene of TbAgo protein, transformed Escherichia coli BL21 (DE3) with the recombinant plasmid to achieve heterologous expression of TbAgo, and then purified by Ni-NTA column to obtain the TbAgo protein produced by the recombinant strain.

The molecular weight of the TbAgo protein obtained by the present invention is about 78.8 kDa, and the enzyme can use 5'-P gDNA to mediate the cutting of single-stranded DNA targets. The optimal reaction temperature range is between 50°C and 75°C; Mn ²⁺ can be used as an active ion, and 250μM-1000μM Mn ²⁺ can keep it highly active; the enzyme has high activity in the NaCl concentration range of 100 mM-500mM; the enzyme has a strong preference for gDNA, and has high activity only when using gDNA modified with a 5' terminal phosphate group, and has low activity when other modifications such as 5'-OH modification are used; the enzyme can use 14nt-35nt 5'-P gDNA to cut at the 10-11 sites of the target DNA; the enzyme can distinguish single-point mismatches between the target DNA and the gDNA.

On the other hand, the present invention provides a method for detecting target nucleic acid based on programmable nuclease TbAgo. This technology not only has the characteristics of high sensitivity, high specificity and high stability, but also can meet the requirements of detecting multiple target nucleic acid molecules in a single reaction system. In addition, this technology also has application prospects in SNV gene detection.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the method for detecting a target nucleic acid molecule according to the present invention.

FIG. 2 is a diagram showing the evolutionary tree of some characterized Argonaute proteins provided in an embodiment of the present invention.

FIG. 3 is a diagram showing a sequence alignment result of some characterized Ago proteins provided in an embodiment of the present invention.

FIG. 4 is a graph showing the purity of TbAgo analyzed by SDS-PAGE gel according to an embodiment of the present invention.

FIG. 5 is a result of measuring the cleavage activity of TbAgo provided in an embodiment of the present invention.

FIG. 6 is a measurement result of the effect of measurement temperature on TbAgo cleavage activity provided in an embodiment of the present invention.

FIG. 7 is a result of determining the effect of different divalent metal ions on the cleavage activity of TbAgo provided in an embodiment of the present invention.

FIG8 is a result of determining the effect of different Mn ²⁺ concentrations on TbAgo cleavage activity provided in an embodiment of the present invention.

FIG. 9 is a result of determining the effect of different NaCl concentrations on TbAgo cleavage activity provided in an embodiment of the present invention.

FIG. 10 is a result of determining the optimal length of guide DNA suitable for TbAgo provided in an embodiment of the present invention.

FIG. 11 is a diagram showing the kinetic results of TbAgo cutting a single-stranded DNA substrate according to an embodiment of the present invention.

FIG12 is a schematic diagram of TbAgo provided in an embodiment of the present invention for single-point mismatches at different sites between gDNA and Target.

FIG. 13 shows that TbAgo provided in an embodiment of the present invention can distinguish the cutting results for single-point mismatches at different sites between gDNA and Target.

FIG. 14 is a result of detecting a target gene using TbAgo enzyme combined with a fluorescent nucleic acid probe provided in an embodiment of the present invention.

FIG. 15 is a result of detecting multiple target genes in a single system using TbAgo enzyme combined with fluorescent nucleic acid probes provided in an embodiment of the present invention.

DETAILED DESCRIPTION

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Acquisition of TbAgo gene sequence

In the NCBI database, similarity search was performed on the known amino acid sequences of TtAgo, and some amino acid sequences with high sequence consistency were selected for analysis using MEGA software to construct a homology evolution tree, and TbAgo was selected as a candidate enzyme. The amino acid sequence of TbAgo is shown in SEQ ID NO:1.

SEQ ID NO:1

MMGDSRSLEVRLNRFLLRPLRPEEEREPWLLVSELNPPPSREDVHALLA LLANRAGGRTARMGDSLLTWSPPESLLLEGTLSWRGNTYTYRLRPLARRVL NPRNPSERDALSALARRLLREVLEQFRREGFWVEGWAFYRKEHARGPGW RVLKGAALDLWVSAEGAMVLEVDPTYRILCDMTLEAWLAQGHPPPKRVKNA YNDRTWELLGLGEEDPQGPGGL NLVEYHASKGRIRDGGWGRVAWVAN PKDAKEKIPHLTSLLIPVLTLEDLHEEGGSNLALSIPWNQRQEETLKVALSVAR RLGVEHPKPVEAKAWRMRMPELRARRRVGKPADALRVGLYRAQETTLALLR LDGGRGWPDFLLKALENAFRASQARLHVREIHADPSQPLAFREALEEAKEA GVQAVLVLTPPLSWEERHRLKALFLKEGLPSQLLNVPIQREERHRLENALLGL LAKAGLQVVALEGAYPADLTVGFDAGGRKSFRFGGAACAVGSDGGHLLWSL PEAQAGERIPGEVVWDLLEEALLVFKRGRLPSRVLLLRDGRLPKDEFTLA LAKLRQLGIGFDLVSVRKSGGGRIYPTRGRLLDGLLVPVEERTFLLLTVHREF RGTPRPLKLVHEEGETPLEA LAEQIYHLTRLYPASGFAFPRLPAPLHLADRLV KEVGRLGVRHLKEVDREKLFFV*

The evolutionary tree result is shown in Figure 2.

Example 2: Alignment of TbAgo with other known pAgo sequences

In this example, multiple sequence alignment of TbAgo with some of the characterized pAgo was performed.

According to literature reports, the targeted cleavage of all pAgo proteins with nuclease activity is mediated by a conserved DEDX (X represents histidine, aspartic acid or asparagine) active center. Through sequence alignment, it can be found that TbAgo has a DEDX active center (Figure 3). Therefore, it is speculated that it may have nuclease activity and needs further in vitro identification and characterization.

Example 3: Heterologous expression and purification of TbAgo protein

The amino acid sequence of TbAgo (WP_071678161.1) was obtained from Example 1. After the gene sequence was synthesized by codon optimization, it was cloned into the pET28a expression vector to obtain the pET28a-TbAgo prokaryotic expression plasmid, which was introduced into E. coli BL21 (DE3) to obtain the pET28a-TbAgo/E. coli BL21 (DE3) prokaryotic expression strain. The expression strain E. coli BL21 (DE3) containing the recombinant plasmid pET28a-TbAgo was inoculated in TB medium containing 50 μg/mL kanamycin, and cultured at 37°C, 220rpm shaking incubator until OD600 was between 2.0-3.0, and IPTG was added at a final concentration of 0.1mM, and cultured at 25°C, 200rpm shaking incubator for 16h-20h to induce the expression of TbAgo protein. The cells were collected by centrifugation, resuspended in a resuspension buffer (containing 20mM Tris-HCl, pH7.5, 0.3M NaCl), and then crushed by high pressure and centrifuged to obtain the supernatant. The protein was affinity purified using a Ni-NTA column, and the eluate was concentrated by ultrafiltration, desalted, and other steps to obtain the purified protein. The purified protein was stored in a buffer containing 20mM Tris-HCl and 0.3M NaCl, and the protein concentration was determined by a BCA kit. The determination steps were carried out according to the operating instructions. Using BSA as a standard, a standard solution was prepared, and a standard curve was drawn. The concentration of the purified target protein was calculated accordingly, and the protein was stored in a -80°C refrigerator for later use. TbAgo protein was analyzed by SDS-PAGE electrophoresis.

The results are shown in Figure 4. The results show that the target protein TbAgo has been purified.

Example 4: TbAgo cleavage activity assay

Design a 40nt single-stranded DNA target nucleic acid molecule modified with a fluorescent group, a 25nt simulated cleavage product single-stranded DNA modified with a fluorescent group, and a 19nt gDNA modified with a 5’ phosphate group or 5’ hydroxyl group, and send them to the company for synthesis.

DNA target nucleic acid molecule sequence (SEQ ID NO: 2):

5’-FAM-aaaataatttaatatactatacaacctactacctcttata-3’

Simulated cleavage product sequence (SEQ ID NO: 3):

5’-FAM-aaaataatttaatatactatacaac-3’

gDNA (SEQ ID NO:4):

5’-HO/P-tgaggtagtaggttgtata-3’

Prepare reaction buffer (containing 10mM Tris-HCl pH7.5, 150mM NaCl), add MnCl ₂ with a final concentration of 0.5mM, 2.5μM TbAgo, 500nM synthetic gDNA and 500nM 5' fluorescent group-modified sequence-complementary single-stranded DNA target nucleic acid molecule to the reaction buffer, react at 75°C for 60min, after the reaction, take 10μL sample, add loading buffer (containing 95% (deionized) formamide, 0.5mmol/L EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol) at a ratio of 1:1, and perform electrophoresis detection under 14% nucleic acid Urea-PAGE.

The results are shown in Figure 5. The results show that TbAgo can use 5'-P gDNA to cut complementary single-stranded DNA.

Example 5: Analysis of TbAgo cleavage characteristics

The cleavage activity of TbAgo was investigated at different temperatures (30°C, 37°C, 50°C, 65°C, 75°C, 85°C, and 95°C). MnCl ₂ with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, 500 nM synthetic 5'-P gDNA, and 500 nM 40 nt 5' fluorescently modified sequence-complementary single-stranded DNA target nucleic acid molecules were added to the reaction buffer. The reaction was carried out at different temperatures for 60 min, and the reaction products were detected by electrophoresis under 14% nucleic acid Urea-PAGE.

The results are shown in Figure 6. The results show that TbAgo can use 5'-P gDNA to cut complementary target nucleic acid molecules in the range of 50°C-75°C.

The concentrations of TbAgo, 5'-P gDNA and target nucleic acid molecules remained unchanged, and CoCl ₂ , CuCl ₂ , MgCl _{2 , MnCl 2 ,} ZnCl ₂ , FeCl ₂ and CaCl ₂ solutions with a final concentration of 0.5 mM were added to the reaction system, respectively. The reaction was carried out at 75°C for 60 min, and electrophoresis was performed under 14% nucleic acid Urea-PAGE to analyze the effect of metal ions on the cleavage activity of TbAgo.

The results are shown in Figure 7. The results show that TbAgo can use Mn ²⁺ , Mg ²⁺ or Co ²⁺ as metal ions to mediate 5'-P gDNA-guided DNA cleavage.

The reaction system, buffer and conditions remained unchanged, and different concentrations of MnCl ₂ were added: 0 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, and 1 mM to determine the optimal MnCl ₂ concentration of TbAgo mediated by 5'-P gDNA.

The results are shown in Figure 8. The results show that 0.25mM-1mM Mn ²⁺ can keep TbAgo highly active.

Adjust the reaction buffer composition to prepare reaction buffers with a final concentration of 10mM Tris-HCl pH7.5 and different concentrations of NaCl (0mM, 50mM, 100mM, 150mM, 250mM, 500mM, 1000mM). Keep other reaction systems unchanged. React at 75℃ for 60min and detect by electrophoresis under 14% nucleic acid Urea-PAGE.

The results are shown in Figure 9. The results showed that TbAgo can exert cleavage activity when the NaCl concentration is 0mM-500mM.

5’-P gDNAs of 10nt-20nt, 25nt, and 35nt were designed (Table 1) to explore the effects of gDNAs of different lengths on the cutting activity of TbAgo.

Table 1

MnCl ₂ with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, 500 nM of synthetic 5'-P gDNA of different lengths and 500 nM of 40 nt 5' fluorescently modified sequence complementary single-stranded DNA target nucleic acid molecules were added to the reaction buffer, and the reaction was carried out at 75°C for 60 min. The reaction products were detected by electrophoresis under 14% nucleic acid Urea-PAGE.

The results are shown in Figure 10. The results show that TbAgo can use 5'-P gDNA with a length of 14nt-35nt to cut complementary target nucleic acid molecules.

MnCl ₂ with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, 500 nM synthetic 19 nt 5'-P gDNA and 500 nM 40 nt 5' fluorescently modified sequence complementary single-stranded DNA target nucleic acid molecules were added to the reaction buffer, and the reaction was carried out at 75°C for 0 min, 3 min, 6 min, 9 min, 12 min, and 15 min, respectively, to measure the cleavage kinetics.

The results are shown in Figure 11. The results show that when the above system is used, TbAgo can basically cut the target DNA completely within 15 minutes.

5'-P gDNA with single base mismatches introduced at positions 1-15 starting from the 5' end of 5'-P gDNA (Figure 12, Table 2), the reaction system remained unchanged, MnCl ₂ , 5'-P gDNA with mismatches at different positions and target DNA were added to determine the effect of TbAgo in distinguishing single base mismatches. The reaction was carried out at 75°C for 60 minutes, and electrophoresis was performed under 14% nucleic acid Urea-PAGE.

Table 2

Sequence Name Sequence (5'-3') SEQ ID NO: gDNA-m1 P-tcaggtagtaggttgtat 17 gDNA-m2 P-tgtggtagtaggttgtat 18 gDNA-m3 P-tgacgtagtaggttgtat 19 gDNA-m4 P-tgagctagtaggttgtat 20 gDNA-m5 P-tgaggaagtaggttgtat twenty one gDNA-m6 P-tgaggttgtaggttgtat twenty two gDNA-m7 P-tgaggtactaggttgtat twenty three gDNA-m8 P-tgaggtagaaggttgtat twenty four gDNA-m9 P-tgaggtagttggttgtat 25 gDNA-m10 P-tgaggtagtacgttgtat 26 gDNA-m11 P-tgaggtagtagcttgtat 27 gDNA-m12 P-tgaggtagtaggatgtat 28 gDNA-m13 P-tgaggtagtaggtagtat 29 gDNA-m14 P-tgaggtagtaggttctat 30 gDNA-m15 P-tgaggtagtaggttgaat 31

The results are shown in Figure 13. The results show that when the mismatch occurs at positions 7-14, and 1, 3, and 5 of the gDNA, the cleavage efficiency of TbAgo is significantly reduced.

Example 6: TbAgo detects a single target nucleic acid molecule

The schematic diagram of the detection method of the target nucleic acid molecule of the present invention is shown in Figure 1. Specifically, the target nucleic acid molecule in the sample to be tested is amplified first; then the corresponding detection system, i.e., Ago protein, gDNA and nucleic acid probe, is added, and the Ago protein will bind to the gDNA and cut the target nucleic acid molecule, thereby generating new gDNA, and the new gDNA will guide the Ago protein to cut the nucleic acid probe; finally, the signal released after the nucleic acid probe is cut is detected.

Using simulated genes 1, 2, and 3 (Gene1, Gene2, and Gene3) as substrates, primers were designed and sent to the company for synthesis. The simulated genes were amplified by PCR using the PrimerSTAR kit of TAKARA, and the specific operation was performed according to the instructions.

According to the sequences of Gene1, Gene2, and Gene3, 5’-P gDNA-gene1, 5’-P gDNA-gene2, and 5’-P gDNA-gene3 were designed and sent to the company for synthesis. The purpose is to mediate TbAgo to cut the amplified target gene fragments, thereby generating a new round of gDNA.

According to the sequence of the new round of gDNA, the fluorescent group and quenching group modified nucleic acid probes reporter-1~3 were designed. Among them, Gene1 was modified with FAM, Gene2 was modified with HEX, and Gene3 was modified with CY5.

The sequences of the simulated genes, primers, gDNA and nucleic acid probes used in this example are shown in Table 3.

Table 3

2 μl of PCR products of three simulated genes or 2 μl of water (negative control) were taken into the reaction buffer, and MnCl ₂ with a final concentration of 0.5 mM, TbAgo with a final concentration of 2.5 μM, the corresponding 250 nM synthetic 5'-P gDNA and 500 nM reporter were added. The reaction was carried out at 75°C for 60 min, and the corresponding fluorescence value was read by a microplate reader.

The results are shown in FIG14 , indicating that the system of the present invention can effectively detect target nucleic acid molecules.

Example 7: Detection of multiple target nucleic acid molecules using a single system using TbAgo

The three pairs of PCR amplification primers in Example 6, namely gene1-F/gene1-R, gene2-F/gene2-R, and gene3-F/gene3-R, were premixed in equal amounts and then PCR amplified the test sample.

Among them, the test sample contains zero, one, two or three target nucleic acid molecules of Gene1, Gene2 and Gene3 respectively.

Take 2 μl of the amplified product in the reaction buffer, add MnCl ₂ with a final concentration of 0.5 mM, TbAgo with a final concentration of 5 μM, synthetic 5'-P gDNA-gene1, 5'-P gDNA-gene2, 5'-P gDNA-gene3 with a final concentration of 250 nM, and reporter1, reporter2, reporter3 with a final concentration of 500 nM, react at 75°C for 60 min, and read the fluorescence values of the corresponding three fluorescent groups with an enzyme reader.

The results are shown in FIG15 , which indicate that the system of the present invention can effectively detect multiple target nucleic acid molecules in a single system.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Sequence Listing

<110> Hangzhou Fustai Biotechnology Co., Ltd.

<120> A TbAgo-based nucleic acid cleavage system and a method and kit for detecting target nucleic acid molecules

<160> 46

<170> SIPOSequenceListing 1.0

<210> 1

<211> 689

<212> PRT

<213> Artificial Sequence

<400> 1

Met Met Gly Asp Ser Arg Ser Leu Glu Val Arg Leu Asn Arg Phe Leu

1 5 10 15

Leu Arg Pro Leu Arg Pro Glu Glu Arg Glu Pro Trp Leu Leu Val Ser

20 25 30

Glu Leu Asn Pro Pro Pro Ser Arg Glu Asp Val His Ala Leu Leu Ala

35 40 45

Leu Leu Ala Asn Arg Ala Gly Gly Arg Thr Ala Arg Met Gly Asp Ser

50 55 60

Leu Leu Thr Trp Ser Pro Pro Glu Ser Leu Leu Leu Glu Gly Thr Leu

65 70 75 80

Ser Trp Arg Gly Asn Thr Tyr Thr Tyr Arg Leu Arg Pro Leu Ala Arg

85 90 95

Arg Val Leu Asn Pro Arg Asn Pro Ser Glu Arg Asp Ala Leu Ser Ala

100 105 110

Leu Ala Arg Arg Leu Leu Arg Glu Val Leu Glu Gln Phe Arg Arg Glu

115 120 125

Gly Phe Trp Val Glu Gly Trp Ala Phe Tyr Arg Lys Glu His Ala Arg

130 135 140

Gly Pro Gly Trp Arg Val Leu Lys Gly Ala Ala Leu Asp Leu Trp Val

145 150 155 160

Ser Ala Glu Gly Ala Met Val Leu Glu Val Asp Pro Thr Tyr Arg Ile

165 170 175

Leu Cys Asp Met Thr Leu Glu Ala Trp Leu Ala Gln Gly His Pro Pro

180 185 190

Pro Lys Arg Val Lys Asn Ala Tyr Asn Asp Arg Thr Trp Glu Leu Leu

195 200 205

Gly Leu Gly Glu Glu Asp Pro Gln Gly Ile Leu Leu Pro Gly Gly Leu

210 215 220

Asn Leu Val Glu Tyr His Ala Ser Lys Gly Arg Ile Arg Asp Gly Gly

225 230 235 240

Trp Gly Arg Val Ala Trp Val Ala Asn Pro Lys Asp Ala Lys Glu Lys

245 250 255

Ile Pro His Leu Thr Ser Leu Leu Ile Pro Val Leu Thr Leu Glu Asp

260 265 270

Leu His Glu Glu Gly Gly Ser Asn Leu Ala Leu Ser Ile Pro Trp Asn

275 280 285

Gln Arg Gln Glu Glu Thr Leu Lys Val Ala Leu Ser Val Ala Arg Arg

290 295 300

Leu Gly Val Glu His Pro Lys Pro Val Glu Ala Lys Ala Trp Arg Met

305 310 315 320

Arg Met Pro Glu Leu Arg Ala Arg Arg Arg Val Gly Lys Pro Ala Asp

325 330 335

Ala Leu Arg Val Gly Leu Tyr Arg Ala Gln Glu Thr Thr Leu Ala Leu

340 345 350

Leu Arg Leu Asp Gly Gly Arg Gly Trp Pro Asp Phe Leu Leu Lys Ala

355 360 365

Leu Glu Asn Ala Phe Arg Ala Ser Gln Ala Arg Leu His Val Arg Glu

370 375 380

Ile His Ala Asp Pro Ser Gln Pro Leu Ala Phe Arg Glu Ala Leu Glu

385 390 395 400

Glu Ala Lys Glu Ala Gly Val Gln Ala Val Leu Val Leu Thr Pro Pro

405 410 415

Leu Ser Trp Glu Glu Arg His Arg Leu Lys Ala Leu Phe Leu Lys Glu

420 425 430

Gly Leu Pro Ser Gln Leu Leu Asn Val Pro Ile Gln Arg Glu Glu Arg

435 440 445

His Arg Leu Glu Asn Ala Leu Leu Gly Leu Leu Ala Lys Ala Gly Leu

450 455 460

Gln Val Val Ala Leu Glu Gly Ala Tyr Pro Ala Asp Leu Thr Val Gly

465 470 475 480

Phe Asp Ala Gly Gly Arg Lys Ser Phe Arg Phe Gly Gly Ala Ala Cys

485 490 495

Ala Val Gly Ser Asp Gly Gly His Leu Leu Trp Ser Leu Pro Glu Ala

500 505 510

Gln Ala Gly Glu Arg Ile Pro Gly Glu Val Val Trp Asp Leu Leu Glu

515 520 525

Glu Ala Leu Leu Val Phe Lys Arg Lys Arg Gly Arg Leu Pro Ser Arg

530 535 540

Val Leu Leu Leu Arg Asp Gly Arg Leu Pro Lys Asp Glu Phe Thr Leu

545 550 555 560

Ala Leu Ala Lys Leu Arg Gln Leu Gly Ile Gly Phe Asp Leu Val Ser

565 570 575

Val Arg Lys Ser Gly Gly Gly Arg Ile Tyr Pro Thr Arg Gly Arg Leu

580 585 590

Leu Asp Gly Leu Leu Val Pro Val Glu Glu Arg Thr Phe Leu Leu Leu

595 600 605

Thr Val His Arg Glu Phe Arg Gly Thr Pro Arg Pro Leu Lys Leu Val

610 615 620

His Glu Glu Gly Glu Thr Pro Leu Glu Ala Leu Ala Glu Gln Ile Tyr

625 630 635 640

His Leu Thr Arg Leu Tyr Pro Ala Ser Gly Phe Ala Phe Pro Arg Leu

645 650 655

Pro Ala Pro Leu His Leu Ala Asp Arg Leu Val Lys Glu Val Gly Arg

660 665 670

Leu Gly Val Arg His Leu Lys Glu Val Asp Arg Glu Lys Leu Phe Phe

675 680 685

Val

<210> 2

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 2

aaaataattt aatatactat acaacctact acctcttata 40

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 3

aaaataattt aatatactat acaac 25

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 4

tgaggtagta ggttgtata 19

<210> 5

<211> 11

<212> DNA

<213> Artificial Sequence

<400> 5

tgaggtagtag 11

<210> 6

<211> 12

<212> DNA

<213> Artificial Sequence

<400> 6

tgaggtagta gg 12

<210> 7

<211> 13

<212> DNA

<213> Artificial Sequence

<400> 7

tgaggtagta ggt 13

<210> 8

<211> 14

<212> DNA

<213> Artificial Sequence

<400> 8

tgaggtagta ggtt 14

<210> 9

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 9

tgaggtagta ggttg 15

<210> 10

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 10

tgaggtagta ggttgt 16

<210> 12

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 12

tgaggtagta ggttgta 17

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 12

tgaggtagta ggttgtat 18

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 13

tgaggtagta ggttgtatag 20

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 14

tgaggtagta ggttgtatag t 21

<210> 15

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 15

tgaggtagta ggttgtatag tatatt 26

<210> 16

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 16

tgaggtagta ggttgtatag tatattaaat tatttt 36

<210> 17

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 17

tcaggtagta ggttgtat 18

<210> 18

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 18

tgtggtagta ggttgtat 18

<210> 19

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 19

tgacgtagta ggttgtat 18

<210> 20

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 20

tgagctagta ggttgtat 18

<210> 21

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 21

tgaggaagta ggttgtat 18

<210> 22

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 22

tgaggttgta ggttgtat 18

<210> 23

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 23

tgaggtacta ggttgtat 18

<210> 24

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 24

tgaggtagaa ggttgtat 18

<210> 25

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 25

tgaggtagtt ggttgtat 18

<210> 26

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 26

tgaggtagta cgttgtat 18

<210> 27

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 27

tgaggtagta gcttgtat 18

<210> 28

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 28

tgaggtagta ggatgtat 18

<210> 29

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 29

tgaggtagta ggtagtat 18

<210> 30

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 30

tgaggtagta ggttctat 18

<210> 31

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 31

tgaggtagta ggttgaat 18

<210> 32

<211> 113

<212> DNA

<213> Artificial Sequence

<400> 32

agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct 60

tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaa 113

<210> 33

<211> 120

<212> DNA

<213> Artificial Sequence

<400> 33

ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg 60

ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc 120

<210> 34

<211> 99

<212> DNA

<213> Artificial Sequence

<400> 34

cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 60

gccgccgcaa ggaatggtgc atgcaaggag atggcgccc 99

<210> 35

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 35

taccgtcttc atggga 16

<210> 36

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 36

tggtttgccc cagcag 16

<210> 37

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 37

tagtaggttg aggccg 16

<210> 38

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 38

agcgcgattt gctggtga 18

<210> 39

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 39

tttcttgatg tctctgacca gaca 24

<210> 40

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 40

ctgattgccc ttcacc 16

<210> 41

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 41

gacagctcat gttatatccc gc 22

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 42

cccttatgcg actcctgcat 20

<210> 43

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 43

gggcgccatc tccttgca 18

<210> 44

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 44

cgcaccctcc catgaagacg gtacggtgcg 30

<210> 45

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 45

cgcacccctg ctggggcaaa ccagggtgcg 30

<210> 46

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 46

cgcaccacgg cctcaaccta ctacggtgcg 30

Claims (8)

1. A nucleic acid cleavage system, comprising:

(a) A programmable endonuclease Ago; the programmable endonuclease Ago is derived from thermophilic bacteria, and the programmable endonuclease Ago is programmable endonuclease TbAgo;

(b) Guide DNA (gDNA); and

(c) A nucleic acid probe, wherein if the nucleic acid probe is cleaved, the cleavage is detectable.

2. The nucleic acid cleavage system according to claim 1, wherein said TbAgo has at least 80% identity with the amino acid sequence as set forth in SEQ ID No. 1.

3. The nucleic acid cleavage system of claim 1, wherein the nucleic acid probe comprises single-stranded DNA with a detectable label.

4. A detection system for a target nucleic acid molecule, characterized in that the detection system comprises a nucleic acid cleavage system according to any one of claims 1, 2, 3.

5. The detection system of claim 4, wherein the target nucleic acid molecule is a pathogenic microorganism, a genetic mutation, or a specific target DNA or RNA.

6. A method for detecting target nucleic acid molecules of non-diagnostic purpose is characterized in that Ago protein, guide DNA, nucleic acid probe and buffer solution are added into a system containing target nucleic acid molecules to be detected, and then the nucleic acid probe is detected.

7. Use of an Ago protein, which is TbAgo or a similar protein having at least 80% identity with the amino acid sequence shown in SEQ ID No. 1, in a method for detecting a target nucleic acid molecule.

8. The detection kit for the target nucleic acid molecules is characterized by comprising Ago protein, guide DNA and a nucleic acid probe, wherein the Ago protein is TbAgo or a similar protein with at least 80% of the same amino acid sequence shown in SEQ ID NO. 1.

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