CN116606839A - Nucleic acid cutting system based on Argonaute protein Tcago and application thereof - Google Patents
Nucleic acid cutting system based on Argonaute protein Tcago and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleic acid cutting system based on Argonaute protein Tcago and application thereof, wherein the nucleic acid cutting system comprises guide DNA, argonaute protein Tcago and a reporter nucleic acid, the amino acid sequence of the Tcago is shown as SEQ ID NO.1 or as a sequence which has at least 85% sequence identity with SEQ ID NO.1 and has the same function, and the nucleic acid cutting system can be applied to the biotechnology fields of nucleic acid detection, enrichment of low-abundance nucleic acid, gene cloning and the like and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a novel high-temperature Argonaute protein Tcago-based nucleic acid cutting system and application thereof.
Background
Argonaute proteins are a family of genes that are present in almost all types of organisms and are very conserved, widely distributed among eubacteria and archaebacteria. Depending on the source, eukaryotic Argonaute proteins (eukaryotic Argonaute proteins, eAgos) and prokaryotic Argonaute proteins (prokaryotic Argonaute proteins, pAgos) can be distinguished. Currently, argonaute proteins of eukaryotic origin are capable of catalyzing RNA-guided RNA cleavage reactions under normal temperature conditions and play a vital role in the RNA interference (RNAi) pathway in vivo. The Argonaute protein of prokaryotic origin is more diverse in function and structure than eAgros, but its physiological function has long been elusive. pAgos plays an important role in host defense and DNA replication, and structural and biochemical studies of some pAgos (mainly from thermophilic bacteria and archaea) have shown that they can exert endonuclease effects in vitro and host defenses in vivo. pAgos can bind to the guide strand of siDNA to specifically cleave and direct the complementarily paired DNA target strand of the strand. pAgos such as Pfago, ttAgo and MjAgo, etc., which have been reported so far, are mainly derived from Gao Wen Suzhu and are used for gene detection.
pAgo nucleases of thermophilic microbial origin with endonuclease activity have been designed for molecular diagnostics and play an important role in viral detection. With the increasing genome of more and more microorganisms, we have urgent need to further mine and biochemically characterize pAgos to understand its basic biological role and explore its potential for use in molecular diagnostics. Early studies focused mainly on high temperature biological sources of pAgos, with the exception that marinitogapizophilia-derived MpAgo favors cleavage of target single stranded DNA (ssDNA) and RNA targets with 5 'end hydroxylated (5' oh) gRNA, and the remaining high temperature sources of pAgos favors cleavage of ssDNA and/or RNA targets with 5 'end phosphorylated (5'P) guide DNA (gDNA). High temperature pAgos has been reported to bind single stranded gDNA or gRNA at high temperatures of 70℃and above, cleave ssDNA targets, and few can cleave RNA targets, which limits the use of high temperature AgoIn vitro application of RNA editing. pAgos from hyperthermia organisms, which have been reported to date, are all preferred Mn 2+ Cleavage of ssDNA target and/or RNA target at Mg 2+ Under conditions, the activity of cleaving ssDNA target and/or RNA target is low, and Mn 2+ Non-specific degradation of nucleic acids can occur under high temperature conditions, which affects the accuracy of high temperature pAgo for nucleic acid detection.
In order to better enrich the tools for manipulation of in vitro gene levels, to understand the physiological functions in pAgos and to further explore the evolutionary process of Argonaute proteins, more in vitro functions of Argonaute proteins need to be characterized, in particular Argonaute proteins with unique properties. Characterization of the in vitro properties of multifunctional or fully functional pAgos is particularly important. With the occurrence of more and more diseases caused by viruses, early diagnosis can effectively prevent further outbreaks of diseases. The high temperature and even the multifunctional pAgos excavation can enrich the existing nucleic acid detection methods and even realize nucleic acid diagnosis independent of PCR and isothermal amplification, which may avoid the disadvantages of the existing nucleic acid detection methods. In addition, the presence of a programmer that cannot tolerate a single base mismatch may solve the problems of false positives and false negatives.
Disclosure of Invention
In view of the above, the invention excavates and characterizes a novel Argonaute protein from high-temperature prokaryote, and the protein can realize the editing of target DNA or target RNA under the mediation of guide DNA, so that the protein can be applied to nucleic acid detection, enrichment of low-abundance nucleic acid, gene cloning and the like, and provides a novel tool for in vitro gene level editing.
The technical scheme of the invention is as follows:
the invention firstly provides a high-temperature Argonaute protein Tcago, which is specifically (A1) or (A2):
(A1) A protein with an amino acid sequence shown as SEQ ID NO. 1;
(A2) A protein having at least 85% sequence identity with the sequence shown in SEQ ID NO.1 and having the same function as the protein of (A1).
Specifically, with respect to the protein (A2), the sequence is an amino acid sequence obtained by substituting, deleting, changing or inserting one or more bases based on the sequence shown in SEQ ID NO.1, or adding 1 to 10 amino acid residues (preferably 1 to 5 amino acid residues; more preferably 1 to 3 amino acid residues) at the N-terminus or C-terminus thereof.
Preferably, the amino acid sequence of the protein of (A2) has at least 90% sequence identity to the sequence shown in SEQ ID No. 1; more preferably, the sequence identity is ≡95%, for example ≡96%, ≡97%, ≡98% or ≡99%.
The invention also provides a biological material related to the high-temperature Argonaute protein Tcago, which specifically comprises the following steps:
(B1) A gene encoding the Argonaute protein TcAgo;
(B2) An expression cassette, vector or cell comprising the gene of (B1);
(B3) A pAgo complex comprising an Argonaute protein TcAgo and single stranded guide DNA.
The gene (B1) has a nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having at least 50% or at least 80% sequence identity with the sequence shown in SEQ ID NO. 2.
Preferably, the gene sequence has at least 90% sequence identity with the sequence shown in SEQ ID NO. 2; more preferably, at least 95% sequence identity.
Wherein the full length of the sequence shown in SEQ ID No.2 is 2400bp,SEQ ID NO.2, the sequence of the gene is mined from the genus Rhizoctonia (Thermogladius calderae), and the encoded proteins are less than 30% identical to PfAgo, mjAgo and TtAgo and are not in the same clade.
The Argonaute protein Tcago can be prepared recombinantly in vitro using the vectors and cells described in (B2). In one embodiment of the invention, a target gene is connected with a pET28a plasmid to obtain a recombinant vector, then the recombinant vector is transformed into escherichia coli to obtain recombinant bacteria, and the recombinant bacteria are induced to express and purified to obtain the target protein.
For the pAgo complex described in (B3), the length of the single-stranded guide DNA is 15 to 30nt, preferably 16 to 21nt, and most preferably 16 to 18nt; the single-stranded guide DNA is a single-stranded DNA molecule (5'P-gDNA) phosphorylated at the 5' end or a single-stranded DNA molecule hydroxylated at the 5' end (5 ' OH-gDNA), preferably a single-stranded DNA molecule phosphorylated at the 5' end.
Based on the high temperature Argonaute protein TcAgo, the invention further provides a nucleic acid cleavage system comprising:
(a) A guide DNA;
(b) The Argonaute protein Tcago;
(c) Optionally a reporter nucleic acid, which cleavage can be detected if it is cleaved by the Argonaute protein TcAgo.
In the above-described nucleic acid cleavage system, the Argonaute protein TcAgo binds to the guide DNA to form a pAgo complex, and when the reporter nucleic acid is contacted with the reporter nucleic acid, the pAgo complex cleaves the reporter nucleic acid at a specific site if the nucleotide sequence complementary to the guide DNA sequence is present in the reporter nucleic acid.
Based on the above nucleic acid cleavage system, the present invention provides a method for specifically cleaving a target nucleic acid, specifically: constructing a nucleic acid cutting system, taking a target nucleic acid as a reporter nucleic acid, wherein the target nucleic acid is target DNA or target RNA, and specifically cutting the target nucleic acid under the mediation of the guide DNA by Argonaute protein Tcago when the target nucleic acid has a nucleotide sequence complementary to the guide DNA.
In the above method for specifically cleaving a target nucleic acid, the target RNA has no higher structure, or has a higher structure, or is double-stranded RNA, or is in vitro transcribed RNA, or is viral genomic RNA, or is mRNA, or other RNA in a cell; the target DNA is ssDNA or double-stranded DNA.
In particular, the data of the examples of the present invention show that when the guide DNA is a 5' -phosphorylated single-stranded DNA molecule, the target nucleic acid may be ssDNA or double-stranded DNA or single-stranded RNA (ssRNA); when the guide DNA is a 5' hydroxylated single-stranded DNA molecule, the target nucleic acid may be ssDNA or double-stranded DNA.
In the above method for specifically cleaving a target nucleic acid, complementary pairing of the target nucleic acid with the guide DNA specifically includes the following two cases:
(C1) The target nucleic acid has a nucleotide sequence that is fully complementary to the guide DNA sequence;
or (C2) the target nucleic acid has a nucleotide sequence with a single base mismatch to the guide DNA sequence.
Specifically, taking a DNA guide of 16-18 nt in length as an example, the single base mismatch in (C2) may have a mismatch site at the 5' end (site 1), a seed region (site 2-8), a central region (site 9-12), a 3' complementary region (site 13-15), and a 3' tail region (site 16-18).
Preferably, the mismatch site has better cleavage specificity in the seed region, the central region or the 3' complementing region; more preferably, the mismatch sites are most specific for cleavage at sites 2, 4-9, 11, 12 and 15.
The example data shows that when any one of the bases at positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 from the 5' end in the region of reverse complementarity of the guide DNA to the target nucleic acid is mismatched with the reporter nucleic acid, the shear rate of the Argonaute protein TcAgo is significantly reduced, which means: under the same reaction conditions, the shear rate of the programmable nuclease Argonaute is reduced by more than or equal to 80%, preferably by more than or equal to 85%, more preferably by more than or equal to 90%.
In the above method for specifically cleaving a target nucleic acid, the reaction temperature at which specific cleavage can occur is 60℃to 100 ℃.
In particular, the example data shows that when the target nucleic acid is a target DNA, the reaction temperature at which specific cleavage occurs may be from 70℃to 100℃and preferably from 90℃to 98℃and optimally from 94℃to 96 ℃; when the target nucleic acid is a target RNA, the reaction temperature at which specific cleavage occurs may be 60℃to 85℃and preferably 70℃to 80℃and optimally 75℃to 80 ℃.
In the above method for specifically cleaving a target nucleic acid, the cleavage system comprises a cleavage site selected from the group consisting of Mg 2+ And Mn of 2+ At least one divalent metal cation of (a), and most preferably Mg 2+ 。
In particular, the example data shows that Mg 2+ At a concentration of at least 1mM and Mg 2+ The higher the concentration of (c) is,the better the cleavage activity of TcAgo; mn (Mn) 2+ At a concentration of up to 5mM, when Mn 2+ At a concentration of 1mM, the cleavage activity of Tcago is best.
In the above method for specifically cleaving a target nucleic acid, naCl may be contained in the cleavage system, and the concentration of NaCl is 0 to 250mM, preferably 50 to 100mM, and most preferably 100 mM.
In the above-described method for specifically cleaving a target nucleic acid, the pH of the cleavage system is preferably 7 to 9.
In the above method for specifically cleaving a target nucleic acid, tcago has a preference for the first base at the 5 '-end of gDNA, and it can be seen from the result of cleavage kinetics that it is more preferred that the first hydroxyl group is thymine (T) or guanine (G) of gDNA, so that the first nucleotide at the 5' -end of gDNA is preferably a phosphorylated modified T or G.
Based on the nucleic acid cleavage system, the invention also provides a method for enriching low-abundance target nucleic acid, which comprises the following steps: constructing a reaction system with a PCR amplification system and the nucleic acid cutting system, wherein the nucleic acid in the system comprises target nucleic acid and non-target nucleic acid, the target nucleic acid is specifically amplified through PCR reaction, and the non-target nucleic acid is specifically cut by Argonaute protein Tcago under the mediation of guide DNA.
The nucleic acid cutting system provided by the invention can also be used in gene detection, and has a very strong application prospect in SNV gene detection due to the fact that the Argonaute protein Tcago in the system has a relatively low tolerance to single-point mismatch between target nucleic acid and gDNA; furthermore, the Argonaute protein Tcago prefers Mg 2+ The example data shows that when the target nucleic acid is single-stranded DNA, 99% of the substrate can be sheared within 10min, and when the target nucleic acid is single-stranded RNA, 60% of the substrate can be sheared within 30 min.
Based on the above nucleic acid cleavage system, the present invention also provides a method for cleaving linear double-stranded DNA or plasmid DNA, specifically comprising: constructing a nucleic acid cutting system, taking linear double-stranded DNA or plasmid DNA in a supercoiled state as a reporter nucleic acid, and performing a cutting reaction to obtain a cut reaction product. In the method, the Argonaute protein Tcago plays a role similar to restriction enzyme, so that the Argonaute protein Tcago is hopeful to be developed into a restriction enzyme tool and has application prospect in the assembly of large-fragment DNA.
In the above method for cutting linear double-stranded DNA, the GC content of 80bp in the vicinity of the cleavage site on the double-stranded DNA is preferably 50% to 70%.
The invention also provides a detection kit which comprises high-temperature Argonaute protein Tcago and can also comprise guide DNA.
On the basis of the high-temperature Argonaute protein Tcago, the nuclease active site of the Tcago shown in SEQ ID NO.1 is mutated to obtain a mutant which completely loses the cutting activity and is marked as Tcago-DM. The mutant can be fused with other effector proteins, and the application direction of Tcago is further expanded.
For example, tcAgo-DM can be used for site-specific targeted blocking of target DNA or target RNA in vitro, in particular: incubating TcAgo-DM with single-stranded gDNA to form a pAgo-guide complex, and contacting the resulting pAgo-guide complex with a target RNA or target DNA, the target having a nucleotide sequence that is largely complementary to the guide sequence, the pAgo-guide complex binding to a region on the target that is largely complementary to the guide.
Compared with the prior art, the invention has the beneficial effects that:
(1) The present invention provides a high temperature Argonaute protein TcAgo, an expression vector comprising a nucleic acid encoding the protein, and compositions, kits and methods for cleaving and editing a target nucleic acid in a sequence-specific manner, and the like. The protein, the nucleic acid, the expression vector, the composition, the kit and the method provided by the invention can carry out site-specific modification on extracellular genetic materials, so that the method can be effectively applied to a plurality of fields of biotechnology, such as nucleic acid detection and gene cloning, and a novel tool is provided for gene cloning and molecular detection of Argonaute protein based on prokaryotic sources.
(2) The high-temperature Tcago provided by the invention has binding activity to single-stranded guide DNA, and has nuclease activity to target DNA or target RNA which is complementarily paired with the single-stranded guide DNA, especially 5'P-gDNA mediated target DNA and target RNA cleavage activity is very high, so that Tcago can be utilized for in-vitro targeted DNA editing or targeted RNA editing.
(3) The Tcago provided by the invention can be in Mg 2+ Under the condition, the DNA has very high nuclease activity on target ssDNA or target RNA which is complementarily paired with the single-stranded guide DNA, and Mn can be effectively avoided 2+ Nonspecific cleavage under high temperature conditions; the invention discovers that the cutting activity prefers Mg for the first time 2+ Therefore, tools for extraction-free nucleic acid detection and for DNA manipulation can be developed using Tcago.
(4) The Tcago provided by the invention can specifically cut DNA and RNA by using a DNA guide, and compared with the RNA, the DNA guide has a short synthetic period and low price, and can greatly save the cost. In addition, the pAgo complex used in the invention does not depend on a special motif near the target site to identify and bind the target, and the nucleic acid guide is convenient to design without considering site restriction.
(5) The Tcago has strong cleavage activity, strictly depends on complementary pairing of a guide and a target to exert the cleavage activity, does not have nonspecific 'collateral cleavage' activity of CRISPR related proteins, and has better specificity.
(6) The Tcago provided by the invention can cut ssDNA by using two gDNA with different modifications (5'P/OH) at the 5' end, the reaction rate of cutting ssDNA is fast, the reaction time is very short, and the reaction time can reach nearly 100% in 5-10 min; tcago has lower requirement on the length of gDNA, and the Tcago has high shearing activity when the gDNA is 16-25 nt.
(7) The Tcago provided by the invention can cut double-stranded DNA (dsDNA) with GC content up to 70% under the guidance of a pair of 5'P-gDNA.
Drawings
FIG. 1 is a diagram showing the result of SDS-PAGE detection for the purity identification of a target protein in example 1;
FIG. 2 is a evolutionary tree of Tcago with partially characterized Argonaute proteins in example 1;
FIG. 3 is a schematic representation of a catalytic DEDX quadruplex of Tcago of example 1 and its sequence alignment with a partially characterized Ago protein;
FIG. 4 is a schematic representation of the sequences of tDNA, tRNA, gDNA and gRNA and the product sequences used in example 2;
FIG. 5 is a graph showing the results of the detection of Tcago and Tcago-DM cleaved target ssDNA and target RNA in example 2;
FIG. 6 is a diagram showing SDS-PAGE detection of products of Tcago in example 3, wherein the products are cleaved from a target nucleic acid under gDNA conditions of different lengths;
FIG. 7 is a diagram showing the SDS-PAGE detection of the products of Tcago cleavage of the target nucleic acid under different metal cations in example 4;
FIG. 8 is a graph of Tcago at various Mn in example 4 2+ Or Mg (Mg) 2+ SDS-PAGE detection result graph of products obtained by cutting target nucleic acid under concentration conditions;
FIG. 9 is a diagram showing SDS-PAGE detection of products of Tcago cleaved from a target nucleic acid under different temperature conditions in example 5;
FIG. 10 is a diagram showing the SDS-PAGE detection of products of Tcago cleaved from a target nucleic acid under different single base mismatch conditions in example 6;
FIG. 11 is a diagram showing the SDS-PAGE detection of the products of the cleavage of the target nucleic acid by Tcago in example 7 under different NaCl concentrations;
FIG. 12 is a graph showing the results of detection of Tcago cleaved double-stranded DNA fragments of different GC contents in example 8.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments.
Unless otherwise indicated, the examples were under routine experimental conditions or under conditions recommended by the manufacturer's instructions. The reagents and materials used, unless otherwise indicated, are commercially available.
Example 1 in vitro expression and purification of Tcago and its mutant Tcago-DM
The nucleotide sequence shown as SEQ ID NO.2 is obtained by amplification from a high-temperature prokaryote of the genus Achillea Thermogladius calderae, the Tcago coding sequence is derived from Thermogladius calderae, and the GenBank number is WP_014737302 after optimization.
The sequence shown in SEQ ID No.2 is connected to pET28a to obtain pET28a-Tcago plasmid, then transformed into escherichia coli BL21 (DE 3), single colony is inoculated into LB liquid medium containing 50mg/mL kanamycin, shake flask culture is carried out on a shaking table at 37 ℃ and 220rpm, and when the bacterial body OD is 600 When 0.8 was reached, the mixture was transferred to an 18℃shaker and IPTG induced overnight. After centrifugation at 6000rpm for 10min to collect the cells, washing with Buffer A (20 mM HEPES Buffer, pH 7.5,250mM NaCl,1mM DTT), the cells were resuspended in Buffer A, PMSF was added at a final concentration of 1mM, and the cells were broken under high pressure and centrifuged at 18000rpm for 30min to collect the supernatant.
After the supernatant was filtered, ni-NTA purification was performed. 10 column volumes (3 additions) were washed with 20mM and 50mM imidazole, 3 column volumes were washed with 100mM, 200mM and 300mM, and samples were taken for SDS-PAGE detection. Collecting the eluted component containing high purity target protein, ultrafiltering and changing to Buffer A. After dilution to 125mM NaCl concentration with 20mM HEPES pH 7.5, heparin column (HiTrap Heparin HP, GE Healthcare) purification was performed. Heparin column was equilibrated with Buffer B (20mM HEPES,pH 7.5,125mM NaCl) beforehand and TcAgo was eluted by increasing the concentration of NaCl. Purified proteins were collected, purified by SDS-polyacrylamide gel assay and activity verification, split into small portions, snap frozen in liquid nitrogen and stored at-80 ℃.
The purified target protein was concentrated at 4℃and 4000rpm using a Millipore 50-kDa ultrafiltration tube, and imidazole was removed by pipetting. The enzyme concentration was quantitatively measured by BCA kit, and the measurement procedure was performed according to the protocol, using BSA as a standard, preparing a standard solution, and drawing a standard curve, thereby calculating the protein concentration of purified TcAgo.
SDS-PAGE identification analysis results are shown in FIG. 1, the expected size of Tcago is 88kDa by http:// www.expasy.org/calculation, and the amino acid sequence of Tcago is shown as SEQ ID NO. 1.
The amino acid sequence and the reported prokaryotic boost are analyzed by MEGAX software to construct a homologous evolutionary tree, and the homologous evolutionary tree is subjected to multiple sequence alignment analysis with the reported high-temperature prokaryotic boost to determine whether the homologous evolutionary tree contains catalytic quadruplex DEDX catalytic residues. The evolutionary tree of TcAgo and partially characterized Argonaute proteins (Ago proteins) is shown in fig. 2, and a schematic diagram of the catalytic dexx quadruplet of TcAgo and its sequence alignment with partially characterized Ago proteins is shown in fig. 3.
FIG. 3 shows the Tcago catalytic quadruplex DEDD at positions 580-591, 617-624, 646-657 and 768-779, respectively, of the sequence shown in SEQ ID NO. 1. The present example further relates to the use of TcAgo catalytic quadruplexes by mutating one or more amino acid residues essential for the catalytic activity of TcAgo proteins to form new nuclease activities: LIIGVDVGHGEV-GLGYPGRETARI-KRILVLRDGRLT-APVHYADKLLDA to A, to give the double mutant Tcago-DM. The mutant was prepared and purified with reference to TcAgo.
EXAMPLE 2 Tcago-based nucleic acid cleavage System
This example constructs a cleavage system comprising TcAgo, guide and target nucleic acid, and activity assays were performed on all possible combinations in order to demonstrate which combinations of guide RNA/DNA and target RNA/DNA can be cleaved by TcAgo. Wherein the sequence diagrams of the target DNA (tDNA), the target RNA (tRNA), the single-stranded gDNA and the single-stranded gRNA are shown in FIG. 4, the arrows indicate predicted cleavage sites, and M1 and M2 are the detection product sequences.
The lysis tests were carried out at 92℃at 4:2:1 (Tcago: guide: target) molar ratio. 800nM Tcago with 400nM guide was placed in a solution containing 10mM HEPES-NaOH, pH 7.5,100mM NaCl,10mM MgCl 2 And 5% glycerol and incubated at 70℃for 10min for guide loading. Nucleic acid targets were added to 200nM. The reaction is carried out at 80℃for 30min (ssRNA as target nucleic acid) or at 92℃for 10min (ssDNA as target nucleic acid). The reaction was stopped by mixing the samples with 2 XTBE-PAGE loading dye (95% formamide, 18mM EDTA,0.025% SDS and 0.025% bromophenol blue) and heating at 95℃for 5min. The lysates were resolved by 20% denaturing PAGE, stained with SYBR Gold (Invitrogen) and visualized with Gel DocTM XR+ (Bio-Rad).
With reference to the nucleic acid cleavage system and reaction procedure of TcAgo, the cleavage activity of TcAgo-DM on target ssDNA and RNA was also determined in this example.
The result of cleavage of the target ssDNA and target RNA by TcAgo is shown in fig. 5. The results show that: (1) no product band (34 nt) was observed in the target DNA/RNA control assay incubated without nucleic acid guide (no guide lane), indicating that product band formation was the result of TcAgo nuclease activity; (2) tcago can cleave tDNA with 5'P-gDNA and 5' OH-gDNA and target RNA with 5'P-gDNA; (3) after mutation of the catalytic tetrad of TcAgo, it will lose all 5'P-gDNA or 5' oh-gDNA activity to cleave the target ssDNA and RNA, i.e. at least one amino acid located in the evolutionarily conserved amino acid tetrad is mutated, which alters the catalytic activity of TcAgo protein, i.e. the tetrad is the key site of TcAgo catalytic activity.
Example 3 Effect of guide Length on cleavage Activity in nucleic acid cleavage System
The DNA molecules of 5'OH or 5'P with the length of 12-40 nt are respectively selected as gDNA, and are incubated and combined with Tcago to form pAgo complex in a cleavage reaction system, and the activity of gDNA with different lengths for recognizing the Tcago and cutting target ssDNA is measured. The reaction system and process are described in example 2.
The measurement results are shown in fig. 6, and the results show that: the length of gDNA has a certain influence on the activity of Tcago in recognizing and cutting target ssDNA, the enzyme can recognize and cut ssDNA by using 15-30 nt gDNA, wherein the effects of 16-25 nt 5'P-gDNA and 16-21nt 5' OH-gDNA are better, and the gDNA length is optimal when 16-18 nt.
Example 4 influence of divalent Metal ion and concentration thereof on target nucleic acid cleavage Activity in nucleic acid cleavage System
In this example, tcago was mixed with 5'P-gDNA in a reaction buffer containing 10mM Tris-Cl pH 8.0, 100mM NaCl,5mM divalent metal cation and 5% glycerol and incubated at 55℃for 10min for guide loading, followed by addition of the target sequence for cleavage activity detection (guide used and target nucleic acid sequence as in example 2). Wherein the divalent metal cation is selected from Mg 2+ 、Mn 2+ 、Fe 2+ 、Co 2+ 、Cu 2+ 、Ni 2+ 、Zn 2+ 、Ca 2+ EDTA-added samples were used as control. The reaction procedure is as described in example 2.
Determination of the influence of different Metal cations on cleavage ActivityAs shown in fig. 7, the results show: the choice of divalent metal cations has a certain influence on the cleavage activity of Tcago, where in Mg 2+ The best cleavage activity under the conditions, next Mn 2 + 。
The minimum concentration of divalent metal ions was further searched for in this example, and Mn was selected at 0.5 mM-20 mM, respectively 2+ Or Mg (Mg) 2+ The cleavage activity of Tcago on the target ssDNA was determined after 10min of reaction at 95℃in the reaction buffer.
The measurement results are shown in fig. 8, and the results show that: the concentration of divalent metal cations has a certain influence on the cleavage activity of Tcago, wherein Mg 2+ At least 1mM, mg 2+ The higher the concentration of TcAgo, the better the cleavage activity of TcAgo; mn (Mn) 2+ At a concentration of up to 5mM, mn 2+ At a concentration of 1mM, the cleavage activity of Tcago is best.
EXAMPLE 5 Effect of reaction temperature on Tcago cleavage target nucleic acid Activity
In this example, tcago was combined with 5'P-gDNA by incubation to form a complex, and then target ssDNA or RNA was added thereto, followed by reaction at 37 to 95℃for 10 minutes (target DNA) or 30 minutes (target RNA), respectively. Other reaction conditions of the reaction system and the guide and target nucleic acid sequences used are the same as in example 2.
After the reaction was completed, 10. Mu.L of 2 XTBE-PAGE loading buffer (1:1) was added to each of the reaction mixtures, and the mixture was heated at 95℃for 5 minutes. 10. Mu.L of the sample was taken and electrophoretically detected in a 20% TBE-PAGE gel, stained with 1 XSYBR Gold nucleic acid dye in the dark for 3-5min, and imaged with an imager.
The results are shown in fig. 9, which shows that: tcago can cut target ssDNA at 70-100 ℃, the cutting activity is relatively high at 90-98 ℃ and the cutting activity is optimal at 94-96 ℃; tcago can cut target RNA at 60-85 deg.C, the cutting activity is relatively high at 70-80 deg.C, and the cutting activity is best at 75-80 deg.C.
EXAMPLE 6 Effect of Single base mispairing of target nucleic acid with gDNA Tcago on cleavage of target nucleic acid Activity
After the gDNA with the Tcago and 5'P different base mismatches is incubated and combined to form a complex, the target ssDNA is added, and the target ssDNA and the guide DNA are subjected to single base mismatch, and the reaction is carried out at 95 ℃ for 10min respectively, so that the cleavage activity is measured. The reaction conditions and target nucleic acid sequence of the reaction system were the same as in example 2, and the guide was a single base mismatch on the guide sequence shown in example 2.
The results of the test are shown in FIG. 10, where m1 indicates a mismatch at position 1, m2 indicates a mismatch at position 2, and so on to position 16, and the results show that: the mismatch occurs in the central region and has the greatest effect on the activity of Tcago, the shearing rate is reduced to below 5 percent, even the mismatch has almost no activity, and particularly, the mismatch occurs in a site 12, namely, a single base mismatch occurs in the central region, and the cutting specificity of Tcago is relatively good.
EXAMPLE 7 Effect of NaCl concentration in nucleic acid cleavage System on target nucleic acid cleavage Activity
This example demonstrates the effect of NaCl concentration on target nucleic acid cleavage activity by placing Tcago with 5'P-gDNA in a solution containing 10mM Tris-Cl pH 8.0, 10mM MgCl 2 And different concentrations of NaCl and 5% glycerol reaction buffer, and at 70 ℃ for 10 minutes incubation for guide loading, then adding the target sequence for cleavage activity detection. The guide and target nucleic acid sequences used in this example are the same as those used in example 2.
And respectively selecting 20-500 mM NaCl and adding the NaCl into the buffer solution, and measuring the cutting activity of Tcago on target ssDNA after reacting for 10min at 95 ℃. The measurement results are shown in fig. 10, and the results show that: the concentration of NaCl has a certain influence on the cleavage activity of Tcago, and the concentration of NaCl may be 20 to 250mM, and among them, 50 to 100mM is preferable, and 100mM is most preferable.
Example 8 cleavage Activity of Tcago on double-stranded Linear DNA of different GC content
In this example, a pair of guide DNAs was designed at 50%, 60% and 70% GC content of the double-stranded linear DNA, and Tcago was combined with forward and reverse 5'P-gDNA cut at 50%, 60% and 70% GC content to form a complex, then 150ng of the target dsDNA fragment was added, respectively, after reaction at 92℃for 10min, 1. Mu.L of proteinase K was added, respectively, at 55℃for 30min, proteinase K was inactivated at 80℃for 10min, and finally 2. Mu.L of 6X Purple loading dye (5:1) was added, followed by mixing. Taking 10 mu L of sample, detecting by electrophoresis on 1% agarose gel, and dyeing for 3-5min by ethidium bromide nucleic acid dye in dark place.
The results after detection are shown in fig. 11, and the results show that: tcAgo can effectively cleave double-stranded DNA linear fragments of different GC content, up to 70% GC content DNA, using a pair of gdnas.
In conclusion, the nuclease Tcago is obtained through in vitro expression and purification and first separation, and the optimal reaction parameters are obtained through a large number of fumbling experiments; moreover, the invention discovers for the first time that Mg can be favored 2+ Can have high-temperature Ago protein with high shearing activity at high temperature, thereby providing a method for enriching low-abundance target nucleic acid and a gene detection method based on a Tcago nucleic acid cutting system. The nucleic acid cutting system has the advantages of non-invasiveness, easiness in operation, rapidness and the like, and can better detect low-abundance mutant genes in liquid biopsy of human. The technology of the invention can be widely applied to various fields of molecular diagnosis related to nucleic acid detection, such as the fields of infectious diseases such as major infectious and pathogen infectious diseases, liquid tumor biopsy, HPV typing detection and the like.
The foregoing is only a part of the embodiments of the present invention, and the scope of the present invention is not limited thereto, and any changes or substitutions that may be easily contemplated by those skilled in the art within the technical scope of the present invention should be included in the scope of the present invention.
Claims (10)
1. A nucleic acid cleavage system based on Argonaute protein TcAgo comprising:
(a) A guide DNA;
(b) The Argonaute protein Tcago;
(c) Optionally a reporter nucleic acid, which cleavage can be detected if it is cleaved by TcAgo;
wherein the amino acid sequence of the Tcago is shown as SEQ ID NO.1 or as a sequence which has at least 85 percent sequence identity with SEQ ID NO.1 and has the same function.
2. The nucleic acid cleavage system based on Argonaute protein Tcago according to claim 1, wherein the guide DNA has a hydroxylation or phosphorylation modification at the 5' end and the guide DNA has a length of 15 to 30nt.
3. A method for specifically cleaving a target nucleic acid, wherein the nucleic acid cleavage system according to claim 1 is constructed, wherein the target nucleic acid is used as a reporter nucleic acid; the target nucleic acid is target DNA or target RNA, and the Tcago specifically cuts the target nucleic acid under the mediation of the guide DNA when the target nucleic acid has a nucleotide sequence complementarily paired with the guide DNA.
4. The method of specifically cleaving a target nucleic acid of claim 3, wherein the complementary pairing comprises: the target nucleic acid has a nucleotide sequence that is fully complementary to the guide DNA sequence, or the target nucleic acid has a nucleotide sequence that has a single base mismatch with the guide DNA sequence.
5. The method for specifically cleaving a target nucleic acid according to claim 3, wherein the reaction temperature for the specific cleavage is 60℃to 100 ℃.
6. The method for specifically cleaving a target nucleic acid according to claim 4, wherein the nucleic acid cleavage system comprises Mg 2+ Or/and Mn 2 。
7. The method according to claim 4, wherein the first nucleotide at the 5' -end of the guide DNA is a phosphorylation-modified thymine or guanine.
8. Use of the nucleic acid cleavage system according to claim 1 or 2 for detection of SNV genes.
9. Use of the nucleic acid cleavage system according to claim 1 or 2 for enriching for low abundance target nucleic acids.
10. Use of a nucleic acid cleavage system according to claim 1 or 2 for cleavage of linear double-stranded DNA or plasmid DNA.
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