CN116024192A - TbAgo-based nucleic acid cleavage system, and detection method and kit for target nucleic acid molecules - Google Patents
TbAgo-based nucleic acid cleavage system, and detection method and kit for target nucleic acid molecules Download PDFInfo
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- CN116024192A CN116024192A CN202111255982.5A CN202111255982A CN116024192A CN 116024192 A CN116024192 A CN 116024192A CN 202111255982 A CN202111255982 A CN 202111255982A CN 116024192 A CN116024192 A CN 116024192A
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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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
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a novel medium-high temperature Argonaute protein TbAgo-based nucleic acid cutting system, and a detection method and a kit of target nucleic acid molecules developed by the novel medium-high temperature Argonaute protein TbAgo-based nucleic acid cutting system.
Background
Nucleic acid detection techniques have been widely used in various fields such as biomolecular medical diagnosis, food safety detection, environmental monitoring, detection of pathogenic microorganisms, genotyping, and the like.
Although conventional nucleic acid detection techniques such as quantitative polymerase chain reaction (qPCR), high throughput sequencing, southern blot, etc. have been widely used, these techniques have drawbacks such as being expensive, time consuming and requiring specialized personnel to operate.
In recent years, a CRISPR/Cas system, i.e., a regularly clustered interval short palindromic repeat and related genes (Clustered regularly interspaced short palindromic repeats/CRISPR associated gene, CRISPR/Cas) are hot spots for research, and nucleic acid detection technologies based on the system are also being developed continuously, such as SHERLOCK, DETECTOR, HOLMES, etc., which are more economical and convenient than traditional nucleic acid detection technologies, but still have certain drawbacks. For example, the Cas protein needs to rely on PAM sites for target nucleic acid molecule recognition and cleavage, so that the sites available for the Cas protein are limited; cas protein can function only under the mediation of guide RNA (gRNA), but RNA has complex production process, high price and is easy to degrade by nuclease in the environment, so that the stability of the whole system is poor; the trans-cleavage activity of Cas proteins, which is excited after recognizing and binding to specific nucleic acids, is a non-specific nuclease activity, so that the nucleic acid detection technology developed based on this technology cannot meet the requirement of detecting multiple target nucleic acid molecules by a single system.
In summary, there is still a need to develop a new nucleic acid detection technology that has an unlimited detection site, is system stable, and can detect multiple target nucleic acid molecules simultaneously.
Argonaute (Ago) proteins were mentioned at the earliest in a study describing Arabidopsis mutants. The Ago proteins reported at present are mainly divided into eukaryotic Ago (eAgo) and prokaryotic Ago (pAgo). eAgo is a key participant in the RNA interference (RNAi) pathway in eukaryotes, and can post-transcriptionally regulate gene expression, thereby defending against invasive RNA viruses and protecting the integrity of the genome. Whereas pAgo can bind to single-stranded guide DNA (gDNA) or guide RNA (gRNA), cleavage of DNA or RNA complementary to the guide sequence at a specific site, i.e., between 10 and 11 from the 5' end of the guide, is considered to be an immune mechanism of a prokaryote as well.
At present only a very small number of pagos are characterized, and most of these Ago proteins are high temperature Ago, which bind single-stranded gDNA or gRNA at high temperature and cleave single-stranded DNA or single-stranded RNA. Compared with a CRISPR/Cas system, pAgo does not depend on PAM sites when cutting a target nucleic acid molecule chain, so that the aim of cutting nucleic acid with any sequence can be achieved through the design of gDNA; gDNA has better stability than gRNA; meanwhile, the cutting action is dependent on the specific sequence of gDNA, so that a plurality of cutting reactions in a single reaction system are not interfered with each other, and the aim of simultaneously detecting a plurality of target nucleic acid molecules in the single system can be fulfilled.
Based on the defects of the existing nucleic acid detection technology and the characteristics of the pAgo system, the development of the novel pAgo protein-based nucleic acid detection technology has important practical significance and wide application prospect.
Disclosure of Invention
In a first aspect of the invention, a nucleic acid cleavage system is provided.
The nucleic acid cleavage system comprises:
(a) A programmable endonuclease Argonaute (Ago); the programmable endonuclease Argonaute is derived from thermophilic bacteria (Thermus brockianus), and the programmable endonuclease Argonaute is a 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 from 50℃to 95℃and preferably from 50℃to 85℃and more preferably from 50℃to 75 ℃.
In another preferred embodiment, the TbAgo includes wild-type TbAgo and mutant TbAgo. The TbAgo has at least 80% identity with the amino acid sequence as set forth in SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of the wild-type TbAgo is as shown in NCBI sequence number wp_ 071678161.1.
In another preferred embodiment, the gDNA is a single stranded DNA molecule phosphorylated at the 5 'end or hydroxylated at the 5' end.
In another preferred embodiment, the gDNA is a single stranded DNA molecule phosphorylated at the 5' end.
In another preferred embodiment, the gDNA has a reverse complementary fragment with the nucleic acid probe, which directs TbAgo to cleave the nucleic acid probe to generate a detectable signal value.
In another preferred embodiment, the gDNA has a length of 10nt to 35nt, more preferably 14nt to 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 assay.
In another preferred embodiment, the nucleic acid probe is a single-stranded DNA molecule having only a 5' -end modified with a fluorescent moiety, which 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 fluorophore comprises: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, texas Red-X, or combinations thereof.
In another preferred embodiment, the quenching group comprises: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.
In another preferred embodiment, the fluorophore and the quencher are each 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 microplate reader.
In another preferred embodiment, the nucleic acid probe is a nucleic acid probe having both a fluorescent group and a biotin group, the fluorescent group being FAM.
In another preferred embodiment, the fluorophore and biotin are each independently located at the 5 'and 3' ends 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 example, 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+ Most preferably Mn 2+ 。
In another preferred embodiment, in the nucleic acid cleavage system, mn 2+ The concentration of (2) is 0. Mu.M-1000. Mu.M, preferably 100. Mu.M-1000. Mu.M, more preferably 250. Mu.M-1000. Mu.M.
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 between 0mM and 1000mM, preferably between 0mM and 500mM, most preferably between 100mM and 250mM.
In another preferred embodiment, when there is a mismatch with the nucleic acid probe in any of the 7 th to 14 th bases and 1 st, 3 rd and 5 th bases from the 5' end in the reverse complement region of the gDNA and nucleic acid probe, the cleavage rate of the programmable endonuclease TbAgo is significantly reduced.
In another preferred embodiment, said significantly reducing the cleavage rate of the programmable endonuclease TbAgo means: under the same reaction conditions, the cleavage rate of the programmable endonuclease TbAgo is reduced by not less than 70%, preferably not less than 80%, and more preferably not less than 90%.
In another preferred embodiment, the concentration of the fluorescent nucleic acid probe in the nucleic acid cleavage system is 0.1. Mu.M-2. Mu.M, preferably 0.5. Mu.M-2. Mu.M, more preferably 0.5. Mu.M-1. Mu.M, most preferably 0.5. Mu.M.
In another preferred embodiment, the concentration of the programmable endonuclease TbAgo in the nucleic acid cleavage system is in the range of 10nM to 10. Mu.M, preferably 500nM to 5. Mu.M, and most preferably 2.5. Mu.M.
In another preferred embodiment, the concentration of gDNA in the nucleic acid cleavage system is 10nM to 10. Mu.M, preferably 100nM to 1. Mu.M, and most preferably 500nM.
In a second aspect of the invention, a detection system for a target nucleic acid molecule is provided.
The detection system comprises a nucleic acid cleavage system according to the first aspect of the invention.
The target nucleic acid molecule is a DNA molecule or an RNA molecule.
In another preferred embodiment, the detection system further comprises a subject of detection, i.e. a target nucleic acid molecule.
In another preferred embodiment, the target nucleic acid molecule in the detection system is derived from a pathogenic microorganism, a genetic mutation, a plant, an animal, a virus, and any combination thereof.
In another preferred embodiment, the detection system can detect the target nucleic acid molecule in the form of N, wherein N is a natural number of 1, 2, 3, 4, 5, etc.
In another preferred embodiment, the target nucleic acid molecule in the detection system comprises a synthetic or natural nucleic acid molecule.
In another preferred embodiment, the target nucleic acid molecule in the detection system comprises a wild-type or mutant nucleic acid molecule.
In another preferred embodiment, the detection system further comprises reagents 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 probes in the detection system comprise one or more different nucleic acid probes.
In another preferred embodiment, the nucleic acid probes in the detection system are in one-to-one correspondence with the target nucleic acid molecules, specifically, sequence complementarity.
In another preferred embodiment, the programmable endonuclease TbAgo recognizes and cleaves the target nucleic acid molecule under the guidance of gDNA, thereby generating a new gDNA, which is complementary in sequence to the corresponding nucleic acid probe.
In another preferred embodiment, the programmable endonuclease TbAgo recognizes and cleaves the corresponding nucleic acid probe under the guidance of the new gDNA, thereby generating a corresponding detectable signal.
In another preferred embodiment, the concentration of the programmable endonuclease TbAgo in the detection system is 10nM to 10. Mu.M, preferably 500nM to 5. Mu.M, most preferably 5. Mu.M.
In another preferred embodiment, the concentration of the target nucleic acid molecule in the detection system is 1fM-200 pM, preferably 1fM-1000fM, most preferably 1fM-100fM.
In another preferred embodiment, the concentration of gDNA in the detection system is 10nM to 10. Mu.M, preferably 100nM to 1. Mu.M, and most preferably 250nM.
In another preferred embodiment, the concentration of nucleic acid probe in the detection system is 100nM to 1000nM, preferably 500nM to 1000nM.
In another preferred embodiment, the detection system further comprises (d) a divalent metal ion, 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 invention.
In another preferred embodiment, the detection system is one wherein the operating temperature of the programmable endonuclease TbAgo is as described in the first aspect of the invention.
In a third aspect of the present invention, there is provided a method for detecting a target nucleic acid molecule of non-diagnostic interest, comprising the steps of:
(a) Providing a sample to be tested;
(b) Providing a reaction system to process a sample to be tested and form a reaction solution; the reaction system is a detection system of a target nucleic acid molecule according to the second aspect of the invention;
(c) The signal of the reaction solution was detected.
Wherein, if the specific signal is detected, the detection sample comprises the specific target nucleic acid molecule; if no specific signal is detected, it is indicated that the test sample does not contain a specific target nucleic acid molecule.
In another preferred embodiment, the reaction system comprises reagents for amplification of a target nucleic acid molecule.
In another preferred embodiment, the nucleic acid amplification method is selected from the group consisting of 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 target nucleic acid molecule is detected by SNP, point mutation, deletion or insertion.
In another preferred embodiment, the signal is read in step (c) using a microplate reader.
In another preferred embodiment, the signal is read in step (c) using a colloidal gold test strip.
In another preferred embodiment, the method of detecting a target nucleic acid molecule is used for in vitro detection.
In another preferred embodiment, the method of detecting a target nucleic acid molecule is non-diagnostic and non-therapeutic.
In a fourth aspect of the invention there is provided the use of an Ago protein in a method of detection of a target nucleic acid molecule, said Ago protein being TbAgo or a similar protein having at least 80% identity to the amino acid sequence shown in SEQ ID No. 1.
In another preferred example, the programmable endonuclease TbAgo comprises TbAgo derived from thermophilic bacteria Thermus brockianus; or a homologous protein having similar cleavage activity.
In another preferred embodiment, the programmable endonuclease TbAgo includes both wild-type and mutant TbAgo.
In another preferred embodiment, the programmable endonuclease TbAgo has an amino acid sequence selected from the group consisting of:
(i) An amino acid sequence as set forth in NCBI sequence No. WP_ 071678161.1; and
(ii) An amino acid sequence obtained by performing substitution, deletion, alteration, or insertion of one or more amino acid residues, or adding 1 to 10 amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues) to the N-terminus or C-terminus thereof based on the sequence shown in NCBI sequence number wp_ 071678161.1; and the amino acid sequence obtained has a sequence identity of ≡80% (preferably ≡90%, more preferably ≡95%, for example ≡96%,. Gtoreq.97%,. Gtoreq.98% or ≡99%) 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, there is provided a detection kit for a target nucleic acid molecule, comprising Ago protein, guide DNA, a nucleic acid probe.
The recombinant plasmid pET28a-TbAgo is constructed after the gene of the TbAgo protein is excavated and the sequences are compared, the recombinant plasmid is transformed into escherichia coli BL21 (DE 3) to realize the heterologous expression of the TbAgo, and the TbAgo protein produced by the recombinant strain is obtained after purification by a Ni-NTA column.
The TbAgo protein obtained by the invention has a molecular weight of about 78.8kDa, and the enzyme can utilize 5' -PgDNA to mediate the cleavage of a single-stranded DNA target. The optimal reaction temperature range is 50-75 ℃; mn can be used 2+ As active ions, 250. Mu.M-1000. Mu.M Mn 2+ Can keep higher activity; the enzyme has higher activity in the concentration range of 100-mM mM NaCl; the enzyme has strong preference on gDNA, has high activity only when gDNA modified by 5 '-terminal phosphate groups is used, and has low activity when other modifications such as 5' -OH modification are performed; the enzyme can cut at the 10-11 sites of target DNA by using 14nt-35nt 5' -P gDNA; the enzyme can distinguish single point mismatches between target DNA and gDNA.
In another aspect, the invention provides a method for detecting a target nucleic acid based on a programmable endonuclease TbAgo. The technology not only has the characteristics of high sensitivity, high specificity and high stability, but also can meet the requirement of detecting multiple target nucleic acid molecules by a single reaction system. In addition, the technology has application prospect in SNV gene detection.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a schematic diagram of a method for detecting a target nucleic acid molecule according to the present invention.
FIG. 2 is a graph of the results of a part of the evolution tree of the characterized Argonaute protein provided by the examples of the present invention.
FIG. 3 is a diagram of the sequence alignment of a partially characterized Ago protein provided by an embodiment of the present invention.
FIG. 4 is a graph showing the purity of TbAgo by SDS-PAGE gel provided in the examples of the present invention.
FIG. 5 is a measurement result of TbAgo cleavage activity according to the example of the present invention.
FIG. 6 is a measurement result of the effect of the measurement temperature on TbAgo cleavage activity provided in the example of the present invention.
FIG. 7 is a graph showing the results of measuring the effect of different divalent metal ions on TbAgo cleavage activity provided in the examples of the present invention.
FIG. 8 is a graph showing the determination of different Mn according to an embodiment of the present invention 2+ Results of the effect of concentration on TbAgo cleavage activity.
FIG. 9 is a graph showing the results of measuring the effect of different NaCl concentrations on TbAgo cleavage activity, provided in the examples of the present invention.
FIG. 10 is a result of determining the optimum length of guide DNA for TbAgo provided by the examples of the present invention.
FIG. 11 is a kinetic result of TbAgo cleavage of single-stranded DNA substrates provided in the examples of the present invention.
FIG. 12 is a schematic of a single point mismatch of TbAgo for different sites between gDNA and Target provided by an embodiment of the invention.
FIG. 13 shows the results of the discrimination of cleavage by TbAgo for single point mismatches at different sites between gDNA and Target provided by the examples of the present invention.
FIG. 14 shows the results of detecting a target gene using a TbAgo enzyme-conjugated fluorescent nucleic acid probe provided in the examples of the present invention.
FIG. 15 is a graph showing the results of detecting multiple target genes in a single system using a TbAgo enzyme-conjugated fluorescent nucleic acid probe provided by 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 of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art without the inventive effort, are intended to be within the scope of the present invention.
Example 1: obtaining of the TbAgo Gene sequence
In NCBI database, similarity search is carried out on the known amino acid sequence of TtAgo, the amino acid sequence with higher partial sequence consistency is selected, MEGA software is adopted for analysis, a homologous evolutionary tree is constructed, and TbAgo is selected as a candidate enzyme. The TbAgo amino acid sequence is shown as SEQ ID NO. 1.
SEQ ID NO:1
MMGDSRSLEVRLNRFLLRPLRPEEREPWLLVSELNPPPSREDVHALLA LLANRAGGRTARMGDSLLTWSPPESLLLEGTLSWRGNTYTYRLRPLARRVL NPRNPSERDALSALARRLLREVLEQFRREGFWVEGWAFYRKEHARGPGW RVLKGAALDLWVSAEGAMVLEVDPTYRILCDMTLEAWLAQGHPPPKRVKNA YNDRTWELLGLGEEDPQGILLPGGLNLVEYHASKGRIRDGGWGRVAWVAN PKDAKEKIPHLTSLLIPVLTLEDLHEEGGSNLALSIPWNQRQEETLKVALSVAR RLGVEHPKPVEAKAWRMRMPELRARRRVGKPADALRVGLYRAQETTLALLR LDGGRGWPDFLLKALENAFRASQARLHVREIHADPSQPLAFREALEEAKEA GVQAVLVLTPPLSWEERHRLKALFLKEGLPSQLLNVPIQREERHRLENALLGL LAKAGLQVVALEGAYPADLTVGFDAGGRKSFRFGGAACAVGSDGGHLLWSL PEAQAGERIPGEVVWDLLEEALLVFKRKRGRLPSRVLLLRDGRLPKDEFTLA LAKLRQLGIGFDLVSVRKSGGGRIYPTRGRLLDGLLVPVEERTFLLLTVHREF RGTPRPLKLVHEEGETPLEALAEQIYHLTRLYPASGFAFPRLPAPLHLADRLV KEVGRLGVRHLKEVDREKLFFV*
The results of the evolutionary tree are shown in figure 2.
Example 2: tbAgo is aligned with other known pAgo sequences
In this example, tbAgo was aligned with partially characterized pAgo in multiple sequences.
It is reported in the literature that the targeted cleavage of all pAgo proteins with nuclease activity is mediated by a conserved active center of DEDX (X stands for histidine, aspartic acid or asparagine). By sequence alignment, the presence of DEDX active centers in TbAgo can be found (FIG. 3). It is therefore speculated that it may have nuclease activity and requires further in vitro characterization.
Example 3: heterologous expression and purification of TbAgo proteins
The amino acid sequence of TbAgo (WP 071678161.1) was obtained from example 1. After the gene sequence is synthesized by codon optimization, cloning the gene sequence into a pET28a expression vector to obtain a pET28a-TbAgo prokaryotic expression plasmid, and introducing the pET28a-TbAgo prokaryotic expression plasmid into E.coli BL21 (DE 3) to obtain a pET28a-TbAgo/E.coli BL21 (DE 3) prokaryotic expression strain. The expression strain E.coli BL21 (DE 3) containing recombinant plasmid pET28a-TbAgo is inoculated into TB medium containing 50 mug/mL kanamycin, shake-cultured at 37 ℃ and 220rpm until the OD is between 600 and 2.0-3.0, IPTG with the final concentration of 0.1mM is added, shake-cultured at 25 ℃ and 200rpm for 16h-20h, and the expression of TbAgo protein is induced. The cells were collected by centrifugation, resuspended in a resuspension buffer (containing 20mM Tris-HCl, pH7.5, 0.3M NaCl), and then the cells were crushed under high pressure, followed by centrifugation to obtain a supernatant. And (3) utilizing Ni-NTA column affinity to purify protein, and carrying out ultrafiltration concentration, desalination and other steps on eluent to obtain purified protein. Purified proteins were stored in a buffer containing 20mM Tris-HCl, 0.3M NaCl, and the protein concentration was measured by BCA kit, and the measurement procedure was performed according to the instructions. BSA is used as a standard substance, a standard solution is prepared, a standard curve is drawn, the concentration of the purified target protein is calculated according to the standard curve, and the protein is stored in a refrigerator at the temperature of minus 80 ℃ for standby. SDS-PAGE electrophoresis analysis of the TbAgo protein.
The results are shown in FIG. 4. The result shows that the target protein TbAgo is purified.
Example 4: tbAgo cleavage Activity assay
A40 nt single-stranded DNA target nucleic acid molecule with a fluorescent group modification, 25nt single-stranded DNA of a simulated cleavage product with a fluorescent group modification, and 19nt gDNA with a 5 'phosphate group or a 5' hydroxyl group modification are designed and sent to a company for synthesis.
DNA target nucleic acid molecule sequence (SEQ ID NO: 2):
5’-FAM-aaaataatttaatatactatacaacctactacctcttata-3’
mock cleavage product sequence (SEQ ID NO: 3):
5’-FAM-aaaataatttaatatactatacaac-3’
gDNA(SEQ ID NO:4)：
5’-HO/P-tgaggtagtaggttgtata-3’
a reaction buffer (containing 10mM Tris-HCl pH7.5, 150mM NaCl) was prepared, and MnCl was added to the reaction buffer at a final concentration of 0.5mM 2 2.5. Mu.M TbAgo,500nM of synthesized gDNA and 500nM of 5' -fluorophore modified sequence complementary single-stranded DNA target nucleic acid molecule were reacted at 75℃for 60min, after the reaction was completed, 10. Mu.L of sample was taken, and a loading buffer (containing 95% (deionized) formamide, 0.5mmol/L EDTA,0.025% bromophenol blue, 0.025% xylene blue) was added in a 1:1 ratio, followed by electrophoresis under 14% nucleic acid Urea-PAGE.
The results are shown in FIG. 5. The results indicate that TbAgo can cleave complementary single stranded DNA using 5' -P gDNA.
Example 5: tbAgo cleavage Property analysis
The cleavage activity of TbAgo was investigated at various temperatures (30 ℃,37 ℃,50 ℃, 65 ℃,75 ℃, 85 ℃, 95 ℃) and MnCl was added to the reaction buffer at a final concentration of 0.5mM 2 TbAgo with a final concentration of 2.5 mu M, 500nM of synthesized 5'-P gDNA and 500nM of 5' -fluorescence modified sequence complementary single-stranded DNA target nucleic acid molecule, reacting at different temperatures for 60min, and performing electrophoresis detection on the reaction product under 14% nucleic acid Urea-PAGE.
The results are shown in FIG. 6. The results indicate that TbAgo can cleave complementary target nucleic acid molecules using 5' -pgdna in the range of 50 ℃ -75 ℃.
TbAgo, 5' -PgDNA and target nucleic acid molecule concentration are unchanged, and CoCl with final concentration of 0.5mM is respectively added into a reaction system 2 、CuCl 2 、MgCl 2 、MnCl 2 、ZnCl 2 、FeCl 2 、CaCl 2 The solution was reacted at 75℃for 60min, and electrophoretically detected under 14% nucleic acid Urea-PAGE, and the effect of metal ions on TbAgo cleavage activity was analyzed.
The results are shown in FIG. 7. The results indicate that TbAgo can utilize Mn 2+ 、Mg 2+ Or Co 2+ As metal ions, 5' -P gDNA-directed DNA cleavage was mediated.
The reaction system, buffer solution and conditions are unchanged, mnCl with different concentrations is added 2 : determination of the optimal MnCl of TbAgo under 5' -PgDNA mediation at 0mM, 0.01mM, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM 2 Concentration.
The results are shown in FIG. 8. The results showed that Mn was 0.25mM-1mM 2+ Can keep the activity of TbAgo higher.
The reaction buffer components were adjusted, and reaction buffers having a final concentration of 10mM Tris-HCl pH7.5 and different concentrations of NaCl (0 mM, 5mM, 100mM, 150mM, 250mM, 500mM, 1000 mM) were prepared, and the other reaction systems were left unchanged, reacted at 75℃for 60 minutes, and then subjected to electrophoresis under 14% nucleic acid Urea-PAGE.
The results are shown in FIG. 9. The results show that TbAgo can exert cleavage activity at NaCl concentrations of 0mM-500 mM.
The 5' -P gDNA of 10nt-20nt and 25nt, 35nt were designed (Table 1), respectively, and the effect of different lengths of gDNA on TbAgo cleavage activity was investigated.
TABLE 1
MnCl with a final concentration of 0.5mM is added to the reaction buffer 2 TbAgo with a final concentration of 2.5 mu M, 5 '-PgDNA with different lengths synthesized at 500nM and 5' -fluorescence-modified sequence complementary single-stranded DNA target nucleic acid molecules at 500nM 40nt, reacting at 75 ℃ for 60min, and performing electrophoresis detection on the reaction product under 14% nucleic acid Urea-PAGE.
The results are shown in FIG. 10. The results indicate that TbAgo can cleave complementary target nucleic acid molecules using 5' -pgdna of 14nt-35nt in length.
MnCl with a final concentration of 0.5mM is added to the reaction buffer 2 The 5 '-PgDNA of 19nt synthesized at a final concentration of 2.5. Mu.M and the 5' -fluorescence-modified sequence complementary single-stranded DNA target nucleic acid molecule of 500nM 40nt were reacted at 75℃for 0min, 3min, 6min, 9min, 12min, 15min, respectively, and cleavage kinetics were determined.
The results are shown in FIG. 11. The results show that with the above system, tbAgo can cut the target DNA substantially completely within 15 min.
Introducing single base mismatch (FIG. 12, table 2) at 1 st to 15 th positions of 5' -PgDNA from 5' -end of 5' -PgDNA, adding MnCl without changing reaction system 2 And 5' -P gDNA and target DNA of different bit mismatch, and determining the effect of TbAgo in distinguishing single base mismatch. The reaction was performed at 75℃for 60min, and the detection was performed by electrophoresis on 14% nucleic acid Urea-PAGE.
TABLE 2
| Sequence name | Sequence (5 '-3') | SEQ ID NO: |
| gDNA-m1 | P- |
17 |
| gDNA-m2 | P- |
18 |
| gDNA-m3 | P- |
19 |
| gDNA-m4 | P- |
20 |
| gDNA-m5 | P-tgaggaagtaggttgtat | 21 |
| gDNA-m6 | P-tgaggttgtaggttgtat | 22 |
| gDNA-m7 | P-tgaggtactaggttgtat | 23 |
| gDNA-m8 | P-tgaggtagaaggttgtat | 24 |
| gDNA-m9 | P- |
25 |
| gDNA-m10 | P-tgaggtagtacgttgtat | 26 |
| gDNA-m11 | P-tgaggtagtagcttgtat | 27 |
| gDNA-m12 | P-tgaggtagtaggatgtat | 28 |
| gDNA-m13 | P-tgaggtagtaggtagtat | 29 |
| gDNA-m14 | P- |
30 |
| gDNA-m15 | P-tgaggtagtaggttgaat | 31 |
The results are shown in FIG. 13. The results show that when mismatches occur at positions 7-14, and positions 1, 3, 5 of gDNA, the cleavage efficiency of TbAgo is significantly reduced.
Example 6: tbAgo for detection of single target nucleic acid molecules
A schematic diagram of the detection method of the target nucleic acid molecule is shown in FIG. 1. Specifically, first amplifying target nucleic acid molecules in a sample to be tested; then adding a corresponding detection system, namely Ago protein, gDNA and a nucleic acid probe, wherein the Ago protein can be combined with the gDNA to cut target nucleic acid molecules so as to generate new gDNA, and the new gDNA can guide the Ago protein to cut the nucleic acid probe; finally, detecting the signal released after the nucleic acid probe is cut.
Primers were designed using mock genes 1, 2 and 3 (Gene 1, gene2 and Gene 3) as substrates, and were synthesized by the company. PCR amplification was performed on the simulated genes using the PrimerSTAR kit from TAKARA, respectively, and the procedure was specifically described.
5' -PgDNA-Gene 1, 5' -PgDNA-Gene 2 and 5' -PgDNA-Gene 3 were designed based on the sequences of Gene1, gene2 and Gene3, respectively, and were synthesized by the company. The aim is to mediate TbAgo to cleave the amplified target gene fragment, thereby generating a new round of gDNA.
And designing nucleic acid probe reporters-1-3 modified by fluorescent groups and quenching groups according to the sequence of the new round of gDNA. Wherein Gene1 is modified by FAM, gene2 is modified by HEX, and Gene3 is modified by CY 5.
The sequence of the mimetic gene, primer, gDNA and nucleic acid probe used in this example is shown in Table 3.
TABLE 3 Table 3
2. Mu.l of PCR products of three mimetic genes or 2. Mu.l of water (negative control) were added to the reaction buffer, respectively, and MnCl was added at a final concentration of 0.5mM 2 TbAgo at a final concentration of 2.5. Mu.M, the corresponding 5' -PgDNA synthesized at 250nM, and 500nM reporter,75 ℃for 60min, and the corresponding fluorescence values were read by an ELISA reader.
The results are shown in FIG. 14, which illustrates 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 with TbAgo
The three pairs of PCR amplification primers of example 6, namely, gene1-F/gene1-R, rgene2-F/gene2-R, gene3-F/gene3-R, were equally premixed and then the PCR amplification was performed on the test sample.
Wherein the test sample contains zero, one, two or three target nucleic acid molecules of Gene1, gene2 and Gene3, respectively.
2 μl of amplified product was added to the reaction buffer, followed by addition of MnCl at a final concentration of 0.5mM 2 TbAgo at a final concentration of 5. Mu.M, 5' -PgDNA-gene 1, 5' -PgDNA-gene 2, 5' -PgDNA-gene 3 synthesized at a final concentration of 250nM, reporter1, reporter2, reporter3 at a final concentration of 500nM, reaction at 75deg.C for 60min, microplate reader readingFluorescence values corresponding to the three fluorophores.
The results are shown in FIG. 15, which illustrates that the system of the present invention can effectively perform detection of multiple target nucleic acid molecules in a single system.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Fusi Tay biotechnology Co., ltd
<120> a TbAgo-based nucleic acid cleavage system, and a method and kit for detecting a target nucleic acid molecule
<160> 46
<170> SIPOSequenceListing 1.0
<210> 1
<211> 689
<212> PRT
<213> Artificial sequence (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 (Artificial Sequence)
<400> 2
aaaataattt aatatactat acaacctact acctcttata 40
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
aaaataattt aatatactat acaac 25
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tgaggtagta ggttgtata 19
<210> 5
<211> 11
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 12
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 13
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
<210> 8
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
<210> 12
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
tgaggtagta ggttgta 17
<210> 12
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
tgaggtagta ggttgtat 18
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
<210> 14
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
tgaggtagta ggttgtatag t 21
<210> 15
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
tgaggtagta ggttgtatag tatatt 26
<210> 16
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tgaggtagta ggttgtatag tatattaaat tatttt 36
<210> 17
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
tcaggtagta ggttgtat 18
<210> 18
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
tgtggtagta ggttgtat 18
<210> 19
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
tgacgtagta ggttgtat 18
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
tgagctagta ggttgtat 18
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tgaggaagta ggttgtat 18
<210> 22
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
tgaggttgta ggttgtat 18
<210> 23
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
tgaggtacta ggttgtat 18
<210> 24
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
<210> 25
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
<210> 26
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
tgaggtagta cgttgtat 18
<210> 27
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
tgaggtagta gcttgtat 18
<210> 28
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
tgaggtagta ggatgtat 18
<210> 29
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
tgaggtagta ggtagtat 18
<210> 30
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
tgaggtagta ggttctat 18
<210> 31
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
tgaggtagta ggttgaat 18
<210> 32
<211> 113
<212> DNA
<213> Artificial sequence (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 (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 (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 (Artificial Sequence)
<400> 35
<210> 36
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
<210> 37
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
<210> 38
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
<210> 39
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
tttcttgatg tctctgacca gaca 24
<210> 40
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
<210> 41
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gacagctcat gttatatccc gc 22
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
<210> 43
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gggcgccatc tccttgca 18
<210> 44
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
cgcaccctcc catgaagacg gtacggtgcg 30
<210> 45
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
cgcacccctg ctggggcaaa ccagggtgcg 30
<210> 46
<211> 30
<212> DNA
<213> Artificial sequence (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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111255982.5A CN116024192A (en) | 2021-10-27 | 2021-10-27 | TbAgo-based nucleic acid cleavage system, and detection method and kit for target nucleic acid molecules |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111255982.5A CN116024192A (en) | 2021-10-27 | 2021-10-27 | TbAgo-based nucleic acid cleavage system, and detection method and kit for target nucleic acid molecules |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116024192A true CN116024192A (en) | 2023-04-28 |
Family
ID=86071087
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111255982.5A Pending CN116024192A (en) | 2021-10-27 | 2021-10-27 | TbAgo-based nucleic acid cleavage system, and detection method and kit for target nucleic acid molecules |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116024192A (en) |
-
2021
- 2021-10-27 CN CN202111255982.5A patent/CN116024192A/en active Pending
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