CN113106087A

CN113106087A - Characterization and application of novel high-temperature Argonaute protein

Info

Publication number: CN113106087A
Application number: CN202110425455.8A
Authority: CN
Inventors: 冯雁; 孙莹璎; 郭翔; 陆慧; 李忠磊
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-04-20
Filing date: 2021-04-20
Publication date: 2021-07-13
Anticipated expiration: 2041-04-20
Also published as: WO2022222920A1; CN113106087B

Abstract

The invention provides characterization and application of a novel high-temperature Argonaute protein. Specifically, the present invention provides a nucleic acid cleavage system, characterized in that the nucleic acid cleavage system comprises: (a) guide dna (gdna); (b) the programmable endonuclease, argonaute (ago); and (c) optionally a reporter nucleic acid, wherein if the reporter nucleic acid is cleaved, the cleavage is detectable. In addition, the invention also provides a system and a method for enriching and detecting low-abundance target nucleic acid based on the programmable endonuclease Ago. The detection system and the detection method provided by the invention can specifically and effectively enrich and efficiently detect low-abundance nucleic acid, and the programmable endonuclease Ago has strong gene manipulation potential.

Description

Characterization and application of novel high-temperature Argonaute protein

Technical Field

The invention belongs to the field of molecular biology and biotechnology, and particularly relates to characterization and application of a novel high-temperature Argonaute protein.

Background

The Argonaute (ago) protein was first mentioned in a study describing mutants of Arabidopsis thaliana. Ago proteins are key players of the eukaryotic RNA interference (RNAi) pathway and can regulate gene expression post-transcriptionally, thereby protecting the host from invading RNA viruses and protecting genomic integrity. Ago proteins reported so far are mainly divided into two categories, eukaryotic Ago (eago) and prokaryotic Ago (pago).

Prokaryotic Ago can bind to single stranded guide DNA or RNA, catalyzing cleavage of target DNA or RNA that is complementarily paired with guide. Unlike CRISPR/Cas systems, prokaryotic Ago does not require a PAM sequence when cleaving a target nucleic acid strand, and can bind to a guide (DNA or RNA) complementary to the target nucleic acid, and cleave at any position of the target.

The high-temperature Ago protein derived from archaea can exert shearing activity at the temperature of more than 70 ℃, and the currently characterized high-temperature Ago can be combined with single-stranded guide DNA or RNA to shear target single-stranded DNA, and a few of the high-temperature Ago proteins can also shear target single-stranded RNA.

Ago protein has also been applied in the field of gene detection in recent years, and can be used for the enrichment of low-abundance mutant DNA in tumor-associated genes, such as TtAgo and PfAgo, which have been reported, due to its property of selective splicing to wild-type and mutant genes. The established technology of 'A-STAR (amplified Specific Target detection)' is an enrichment technology which has high specificity and can be combined with a multi-terminal detection technology. Because of the limitations of the current clinical rare mutation detection methods, the research on novel detection technologies and novel nucleic acid tool enzymes is becoming a research hotspot.

However, some current detection methods using nucleic acid tool enzymes still have the disadvantages of high detection cost and complicated steps.

Therefore, there is an urgent need in the art to develop a more efficient method for enriching and detecting low-abundance DNA.

Disclosure of Invention

The invention aims to provide a more efficient low-abundance DNA enrichment and detection method.

In a first aspect of the present invention, there is provided a nucleic acid cleavage system comprising:

(a) guide dna (gdna);

(b) the programmable endonuclease, argonaute (ago); and

(c) optionally a reporter nucleic acid, wherein if said reporter nucleic acid is cleaved, said cleavage is detectable.

In another preferred embodiment, the temperature of the nucleic acid cleavage system is 80 to 99.9 ℃, preferably 90 to 99.9 ℃, more preferably 94 to 96 ℃, and still more preferably 95 ℃.

In another preferred example, the programmable endonuclease Argonaute is derived from the prokaryote Thermococcus eurythermalis (Thermococcus eurythermalis), Methanopyrus jannaschii (Methanopyrus portorcus), Methanococcus jannaschii (Methanopyrus jannaschii), Pyrococcus furiosus (Pyrococcus furiosus), a genus of Thermotogaceae (Marinicola piphilus), Aquifex aeolicus (Aquifex aeolicus), Clostridium butyricum (Clostridium butyricum), Clostridium perfringens (Clostridium perfringens), Thermus thermophilus (Thermus thermophilus), Bacillus halophilus gracilis (Natronobacterium gregorgyi), Enterobacter intestinalis (Enterobacter barrettii), Mariotis mosaic (Kurthia malaysicus), Synechococcus elongatus (Synechococcus macrococcus).

In another preferred embodiment, the programmable endonuclease Argonaute is derived from thermophilic bacteria (Thermococcus eurythermalis), and the programmable endonuclease Argonaute is a programmable endonuclease TeAgo.

In another preferred example, the TeAgo comprises wild-type and mutant TeAgo.

In another preferred example, the amino acid sequence of the wild-type programmable endonuclease TeAgo is shown as NCBI sequence No. WP _ 050002102.1.

In another preferred embodiment, the guide DNA is 5 'phosphorylated and 5' hydroxylated, 5 'provided with a Biotin group and 5' provided with NH₂C₆The radical, 5 'having a FAM group or 5' having an SHC group₆A single-stranded DNA molecule of a group.

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

In another preferred embodiment, the guide DNA has a reverse complementary fragment to the reporter nucleic acid.

In another preferred embodiment, the guide DNA has a length of 5 to 30nt, more preferably 15 to 21nt, and most preferably 16 to 18 nt.

In another preferred embodiment, the nucleotide sequence of the guide DNA is shown in SEQ ID NO. 23.

In another preferred embodiment, the reporter nucleic acid is single-stranded dna (ssdna).

In another preferred embodiment, the endonuclease, Argonaute, is capable of cleaving at the site of the reporter nucleic acid bound to position 10-11 from the 5' end of the gDNA.

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

In another preferred embodiment, the electrophoresis is a 16% nucleic acid Urea-PAG electrophoresis detection method.

In another preferred embodiment, the reporter nucleic acid is a fluorescent reporter nucleic acid with a fluorophore and/or a quencher.

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

In another preferred embodiment, the fluorescent group and the quencher group are located on both sides of the complementary region of the fluorescent reporter nucleic acid and the guide DNA, respectively.

In another preferred embodiment, the fluorescent reporter nucleic acid is 10 to 100nt, preferably 20 to 70nt, more preferably 30 to 60nt, more preferably 40 to 50nt, most preferably 45nt in length.

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

In another preferred embodiment, the quenching group comprises: BHQ, TAMRA, DABCYL, DDQ, or combinations thereof.

In another preferred embodiment, the fluorescent reporter nucleic acid is a single-stranded DNA molecule having only a fluorophore, which is FAM.

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

In another preferred embodiment, the divalent metal ion is Mn²⁺。

In another preferred embodiment, the concentration of the divalent metal ion in the nucleic acid cleavage system is 10. mu.M-3 mM, preferably 50. mu.M-2 mM, more preferably 100. mu.M-2 mM.

In another preferred embodiment, the nucleic acid cleavage system further comprises: (e) and (4) a buffer solution.

In another preferred embodiment, the concentration of NaCl in the buffer is 500mM or less, preferably 20 to 500 mM.

In another preferred embodiment, the pH of the buffer is 7-9, preferably 8.0.

In another preferred embodiment, the cleavage rate of the programmable endonuclease, Argonaute, is significantly reduced when a mismatch between any of the bases from position 4 to position 12 from the 5' end, and positions 14 to 15, is present in the reverse complementary region of the gDNA and the reporter nucleic acid.

In another preferred embodiment, said significantly reducing the cleavage rate of the programmable endonuclease Argonaute is: under the same reaction condition, the shearing rate of the programmable endonuclease Argonaute is reduced by more than or equal to 80 percent, preferably reduced by more than or equal to 85 percent, and more preferably reduced by more than or equal to 90 percent.

In another preferred embodiment, the guide DNA is complementary to the sequence of the fluorescent reporter nucleic acid and directs the TeAgo enzyme to cleave the fluorescent reporter nucleic acid, thereby generating a detectable signal (e.g., fluorescence).

In another preferred embodiment, the concentration of the fluorescent reporter nucleic acid in the nucleic acid cleavage system is 0.4. mu.M-4. mu.M, preferably 0.6. mu.M-2. mu.M, more preferably 0.8. mu.M-1. mu.M, and most preferably 0.8. mu.M.

In another preferred embodiment, the concentration of the programmable endonuclease Ago is 10nM to 10. mu.M, preferably 100nM to 1. mu.M, more preferably 300nM to 500nM, and most preferably 400nM in the nucleic acid cleavage system.

In another preferred embodiment, the concentration of the guide DNA in the nucleic acid cleavage system is 10nM to 10. mu.M, preferably 100nM to 3. mu.M, more preferably 1. mu.M to 2.5. mu.M, and most preferably 2. mu.M.

In a second aspect of the present invention, a reaction system for enriching low-abundance target nucleic acid is provided, wherein the reaction system is used for simultaneously performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction on a nucleic acid sample, thereby obtaining an amplification-cleavage reaction product;

wherein the nucleic acid sample comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid;

the nucleic acid cleavage reaction is used for specifically cleaving non-target nucleic acids but not cleaving the target nucleic acids;

the amplification-cleavage reaction system comprises (i) reagents required to carry out a PCR reaction and (ii) a nucleic acid cleavage system according to the first aspect of the invention.

In another preferred embodiment, the concentration of the programmable endonuclease Argonaute (ago) in the reaction system is 20 to 200nM, preferably 30 to 150nM, and more preferably 40 to 100 nM.

In another preferred embodiment, the reagents required to perform the PCR reaction include PCR Taq Master Mix (available from abm (Applied Biological Materials (abm) Inc.)).

In another preferred embodiment, the reagents required for performing the PCR reaction further comprise amplification primer pairs for the target nucleic acid.

In another preferred embodiment, the concentration of each primer in the amplification primer pair of the target nucleic acid is 100-300nM, preferably 150-250nM, and more preferably 200 nM.

In another preferred embodiment, the concentration of the target nucleic acid is 0.5-5nM, preferably 0.8-2nM, more preferably 1 nM.

In another preferred embodiment, the gDNA comprises forward gDNA and reverse gDNA;

wherein, the forward gDNA is gDNA having the same sequence segment as the target nucleic acid, and the reverse gDNA is gDNA having a reverse complementary sequence segment with the target nucleic acid.

In another preferred embodiment, the reaction system further comprises: a divalent metal ion.

In another preferred embodiment, the divalent metal ion is Mn²⁺。

In another preferred embodiment, the concentration of the divalent metal ion in the reaction system is 50mM-2M, preferably 100mM-1M, and more preferably 0.5 mM.

In another preferred example, the reaction temperature (reaction procedure) of the reaction system is: 94 ℃ for 5 min; the number of cycles is 10-30(94 ℃, 30 s; 52 ℃, 30 s; 72 ℃, 20 s); 72 ℃ for 1 min.

In another preferred embodiment, the target nucleic acid is selected from the group consisting of: a wild type EGFR sequence fragment, an EGFR E746-A750 mutant sequence fragment, and an EGFR L858R mutant sequence fragment.

In another preferred embodiment, the nucleotide sequence of the wild-type EGFR sequence fragment is shown in SEQ ID NO. 1, and the nucleotide sequences of the amplification primer pair are shown in SEQ ID NO. 3 and 4, respectively.

In another preferred embodiment, the nucleotide sequence of the EGFR E746-A750 mutant sequence fragment is shown as SEQ ID NO. 2, and the nucleotide sequences of the amplification primer pairs are shown as SEQ ID NO. 5 and 6, respectively.

In another preferred embodiment, the nucleotide sequence of the wild-type EGFR sequence fragment is shown in SEQ ID NO. 11, and the nucleotide sequences of the amplification primer pairs are shown in SEQ ID NO. 13 and 14, respectively.

In another preferred embodiment, the nucleotide sequence of the EGFR L858R mutant sequence fragment is shown as SEQ ID NO. 12, and the nucleotide sequences of the amplification primer pairs are shown as SEQ ID NO. 15 and 16, respectively.

In a third aspect of the present invention, there is provided a method for enriching a low abundance target nucleic acid, comprising the steps of:

(a) providing a nucleic acid sample containing a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid,

and the abundance of the target nucleic acid in the nucleic acid sample is F1 a;

(b) performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction in an amplification-cleavage reaction system by taking the nucleic acid in the nucleic acid sample as a template, thereby obtaining an amplification-cleavage reaction product;

wherein the nucleic acid cleavage reaction is used to specifically cleave non-target nucleic acids but not the nucleic acid of interest;

and wherein the amplification-cleavage reaction system comprises (i) reagents required to carry out a PCR reaction and (ii) a nucleic acid cleavage system according to the first aspect of the invention;

wherein the abundance of the target nucleic acid in the amplification-cleavage reaction product is F1b,

wherein the ratio of F1b/F1a is not less than 10.

In another preferred embodiment, the target nucleic acid and the non-target nucleic acid differ by only one base.

In another preferred embodiment, the ratio of F1b/F1a is not less than 10 when F1a is not less than 1% and not more than 10%, the ratio of F1b/F1a is not less than 100 when F1a is not less than 0.5%, and the ratio of F1b/F1a is not less than 200 when F1a is not more than 0.1%.

In another preferred embodiment, the nucleic acid sample includes a directly heat-cleaved nucleic acid sample, a directly lyase protease-treated nucleic acid sample, an extracted nucleic acid sample, a PCR pre-amplified nucleic acid sample, or any nucleic acid-containing sample.

In another preferred embodiment, the PCR-preamplified nucleic acid sample is PCR amplification products obtained by 1-30 cycles, preferably 10-20 cycles, and more preferably 15-30 cycles.

In another preferred embodiment, the target nucleic acid is a nucleotide sequence containing mutations.

In another preferred embodiment, the mutation is selected from the group consisting of: insertions, deletions, substitutions of nucleotides, or combinations thereof.

In another preferred embodiment, the non-target nucleic acid (or second nucleic acid) is a wild-type nucleotide sequence, a highly abundant nucleotide sequence, or a combination thereof.

In another preferred embodiment, the abundance of said non-target nucleic acid in said nucleic acid sample is F2 a.

In another preferred example, F1a + F2a is 100%.

In another preferred embodiment, the ratio F2a/F1a is greater than or equal to 20, preferably greater than or equal to 50, more preferably greater than or equal to 100, most preferably greater than or equal to 1000 or greater than or equal to 5000.

In another preferred embodiment, the abundance of said non-target nucleic acid in said amplification-cleavage reaction product is F2 b.

In another preferred example, F1b + F2b is 100%.

In another preferred embodiment, F1b/F2b is 0.5 or more, preferably 1 or more, more preferably 2 or more, most preferably 3 or 5 or more.

In another preferred embodiment, the ratio F1b/F1a is 200 or more, preferably 500 or more, more preferably 1000 or more, most preferably 2000 or more or 5000 or more.

In another preferred embodiment, F1a is 0.5% or less, preferably 0.2% or less, more preferably 0.1% or less, and most preferably 0.01% or less.

In another preferred embodiment, F1b is 10% or more, preferably 30% or more, more preferably 50% or more, most preferably 70% or less.

In another preferred embodiment, the "reagents required for performing PCR reaction" include: a DNA polymerase.

In another preferred embodiment, the "reagents required for performing PCR reaction" further include: dNTP, 1-5Mm Mg²⁺And PCR buffer solution.

In another preferred embodiment, the gDNA in the nucleic acid cleavage system forms a first complementary binding region with the nucleic acid sequence of the targeted region of the target nucleic acid (i.e., the first nucleic acid); and the gDNA in the nucleic acid cleavage system also forms a second complementary binding region with the nucleic acid sequence of the targeted region of the non-target nucleic acid (i.e., the second nucleic acid).

In another preferred embodiment, there are at least 2 mismatched base pairs in the first complementary binding region.

In another preferred embodiment, there are 0 or 1 mismatched base pairs in the second complementary binding region.

In another preferred embodiment, there are 1 mismatched base pairs in the second complementary binding region.

In another preferred embodiment, there are at least 2 mismatched base pairs in the first complementary binding region, resulting in the complex not cleaving the target nucleic acid; and 1 mismatched base pair in the second complementary binding region, thereby causing the complex to cleave the non-target nucleic acid.

In another preferred embodiment, the targeting region of the target nucleic acid (i.e., the first nucleic acid) corresponds to the targeting region of a non-target nucleic acid (i.e., the second nucleic acid).

In another preferred embodiment, in the amplification-cleavage reaction system, the nucleic acid cleavage enzyme is 30nM, and the DNA polymerase is a thermostable polymerase, preferably Taq DNA polymerase, LA Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Phusion DNA polymerase, KOD DNA polymerase, etc., more preferably 2X PCR precision TMMaster Mix.

In another preferred example, the amount of the nucleic acid as a template in the amplification-cleavage reaction system is 0.1 to 100 nM.

In another preferred example, the method further comprises:

(c) detecting the amplification-cleavage reaction product, thereby determining the presence, absence and/or amount of the target nucleic acid.

In another preferred embodiment, the detection in step (c) comprises quantitative detection, qualitative detection, or a combination thereof.

In another preferred embodiment, the quantitative determination is selected from the group consisting of: TaqMan fluorescent quantitative PCR, Sanger sequencing, q-PCR, ddPCR, chemiluminescence method, high-resolution melting curve method, NGS and the like; preferably selected from TaqMan fluorescent quantitative PCR, Sanger sequencing.

In another preferred embodiment, the first nucleic acid comprises n different nucleic acid sequences, wherein n is a positive integer ≧ 1.

In another preferred example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more.

In another preferred embodiment, n is 2 to 1000, preferably 3 to 100, more preferably 3 to 50.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, said nucleic acid sample comprises nucleic acids from a test sample, wherein said test sample is selected from the group consisting of: blood, cells, serum, saliva, body fluids, plasma, urine, prostatic fluid, bronchial lavage, cerebrospinal fluid, gastric fluid, bile, lymph fluid, peritoneal fluid, stool, and the like, or combinations thereof.

In another preferred embodiment, the low-abundance target nucleic acid is selected from the group consisting of: a wild type EGFR sequence fragment, an EGFR E746-A750 mutant sequence fragment, and an EGFR L858R mutant sequence fragment.

In another preferred example, when the low-abundance target nucleic acid is a wild-type EGFR sequence fragment or an EGFR E746-A750 mutant sequence fragment, the primer sequences in the TaqMan fluorescence quantitative PCR method are respectively shown as

SEQ ID NO

9 and 10;

and the probe sequence for detecting the wild type EGFR sequence fragment is shown as SEQ ID NO. 7, and the probe sequence for detecting the EGFR E746-A750 mutant sequence fragment is shown as SEQ ID NO. 8.

In another preferred example, when the low-abundance target nucleic acid is a wild-type EGFR sequence fragment or an EGFR L858R mutant sequence fragment, the primer sequences in the TaqMan fluorescence quantitative PCR method are respectively shown as SEQ ID NO. 19 and 20;

and the probe sequence for detecting the wild type EGFR sequence fragment is shown as SEQ ID NO. 17, and the probe sequence for detecting the EGFR L858R mutant sequence fragment is shown as SEQ ID NO. 18.

In a fourth aspect of the invention, there is provided a kit for detecting a target nucleic acid molecule, the kit comprising:

(i) the reaction system for enriching low-abundance target nucleic acid or the reagent for preparing the reaction system according to the second aspect of the present invention;

(ii) a detection reagent for detecting a low-abundance target nucleic acid; and

(ii) instructions for use which describe the method according to the third aspect of the invention.

In another preferred embodiment, the kit comprises:

(a) a first container and a guide DNA located in the first container;

(b) a second container and a programmable endonuclease, argonaute (ago), located in the second container; and

(c) a third container and a reagent for nucleic acid amplification reaction in the third container.

In another preferred embodiment, the kit further comprises:

(d) a fourth container and a detection reagent of low-abundance target nucleic acid positioned in the fourth container.

In another preferred embodiment, the reagent for detecting a low-abundance target nucleic acid comprises: primers, probes, and the like.

In another preferred embodiment, the reagent for detecting a low-abundance target nucleic acid comprises: primers and probes required for TaqMan fluorescent quantitative PCR, or reagents required for Sanger sequencing.

In another preferred embodiment, the kit further comprises:

(e) a fifth container and a divalent metal ion located in the fifth container.

In another preferred embodiment, the kit further comprises:

(f) a sixth container and a buffer solution in the sixth container.

In another preferred example, the first container, the second container, the third container, the fourth container, the fifth container and the sixth container may be the same or different containers.

In the fifth aspect of the invention, the application of the programmable endonuclease Argonaute is provided, and the application is used for preparing a reagent or a kit for detecting a target molecule or a reagent or a kit for detecting low-abundance target nucleic acid.

In another preferred embodiment, the programmable endonuclease, Argonaute, is derived from thermophilic bacteria (Thermococcus eurythermalis); or a homologous analogue thereof having the same or similar function.

In another preferred example, the TeAgo comprises wild-type and mutant TeAgo.

In another preferred embodiment, the programmable endonuclease, Argonaute, has an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as set forth in NCBI sequence No. WP _ 050002102.1; 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) at the N-terminus or C-terminus thereof on the basis of the sequence shown as NCBI sequence No. WP _ 050002102.1; and the obtained amino acid sequence has a sequence identity of more than or equal to 85% (preferably more than or equal to 90%, more preferably more than or equal to 95%, such as more than or equal to 96%, more than or equal to 97%, more than or equal to 98% or more than or equal to 99%) with the sequence shown as NCBI sequence No. WP _ 050002102.1; and the obtained amino acid sequence has the same or similar functions as (i).

The invention aims to provide a novel high-temperature nuclease with multiple guide strand and substrate strand shearing preference.

In order to achieve the above object, the present invention provides a high temperature Argonaute-TeAgo protein having high temperature nuclease activity, which is a starting strain of a thermophilic bacterium Thermococcus eurythermalis isolated from deep sea thermodrainage port (depth 2006.9 m) of guaima basin.

In another aspect, the present invention provides a gene of a high temperature Argonaute, TeAgo protein, encoding the protein of the high temperature nuclease TeAgo as described above.

According to the invention, after the gene of the TeAgo protein is excavated and the sequence of the gene is compared, a recombinant plasmid pET28 a-TeAgo is constructed, the recombinant plasmid is transformed into escherichia coli (DE3), the heterologous expression of the TeAgo is realized, and then the Ago protein produced by the recombinant strain is obtained through the purification of a Ni-NTA column.

The molecular weight of the novel high-temperature Ago protein obtained by the invention is about 88kDa, the enzyme can simultaneously utilize 5 ' -phosphorylated gDNA and 5 ' -hydroxylated gDNA to mediate the shearing of single-stranded DNA and single-stranded RNA target nucleic acid, and the shearing efficiency of the 5 ' -phosphorylated gDNA mediated single-stranded DNA target nucleic acid is highest. The optimal reaction temperature range is between 90 and 99.9 ℃, and the thermal stability at 95 ℃ is good; can utilize Mn²⁺As active ion, Mn 50-2000. mu.M²⁺Can keep higher activity; the activity of the enzyme is higher when the NaCl concentration range is 20-500 mM; the enzyme can utilize 15nt-21nt of 5' -P gDNA to generate a classical shearing product at 10-11 sites; the enzyme has strong preference for gDNA, has high activity only when gDNA modified by 5 ' -end 5 ' -P, and other modifications such as 5 ' -OH, 5 ' -Biotin, and 5 ' -NH₂5 '-FAM, 5' -SH and the like are low in activity; the enzyme can distinguish single-point mismatch and double-point mismatch between target and gDNA, and has certain application prospect in SNV gene detection. The enzyme can effectively enrich EGFR del E746-A750 and EGFR L858R mutant genes by using an A-STAR detection technology and a PCR-cleavage coupling reaction.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 shows the results of phylogenetic analysis for TeAgo.

FIG. 2 shows the results of SDS-PAGE analyzing the TeAgo protein. Wherein, the lanes from left to right are respectively protein Marker, thallus cracking supernatant, thallus cracking sediment and purified TeAgo.

FIG. 3 shows the results of shear activity assay of TeAgo.

Fig. 4 shows a graph of the results for the optimal temperature range required for the TeAgo reaction.

FIG. 5 shows a graph of the results of thermal stability of TeAgo at 90 deg.C (A) and 95 deg.C (B).

FIG. 6 is a graph showing the results of the effect of divalent metal ion type (A) and concentration (B) on the shear activity of TeAgo.

FIG. 7 shows the effect of the length of 5' -phosphorylated gDNA on the TeAgo cleavage activity.

FIG. 8 shows the results of the temperature range of NaCl that TeAgo can tolerate.

FIG. 9 is a graph showing the results of the preference of TeAgo for the 5' end modification of gDNA.

FIG. 10 shows a graph of the results of differential cleavage by TeAgo against a single point mismatch at different sites between gDNA and Target.

Wherein, A shows the design method and specific sequence of gDNA in single-point mismatch experiment; b shows the experimental results of single point mismatches.

FIG. 11 shows the results of differential cleavage by TeAgo for a double-site mismatch at positions 10-11 between gDNA and Target.

Wherein, A shows the design method and specific sequence of gDNA in double-point mismatch experiment; b shows the results of the double-point mismatch experiments.

FIG. 12 shows a standard curve for the detection of EGFR del E746-A750 low abundance mutant DNA substrate by the double TaqMan probe method.

FIG. 13 shows the results of high sensitivity detection and preferential enrichment of EGFR-delE746-A750 low abundance mutant DNA (5%, 1%, 0.1%) substrate by TeAgo.

FIG. 14 shows a standard curve for the dual TaqMan probe method for detection of EGFR L858R low abundance mutant DNA substrate.

FIG. 15 shows the results of high sensitivity detection and preferred enrichment of low abundance mutant DNA (5%, 1%,) substrate from EGFR L858R by Teago.

Detailed Description

The inventor develops a method for enriching and detecting low-abundance mutant DNA with high sensitivity, good specificity and high flux for the first time through extensive and intensive research and a large amount of screening. Specifically, the inventor obtains the nuclease TeAgo through in vitro expression and purification and separation, and obtains the optimal reaction parameters thereof through a large number of groping experiments, thereby providing a method for enriching low-abundance target nucleic acid based on TeAgo and a corresponding detection method. The invention has the advantages of non-invasiveness, easy operation, rapidness and the like, the sensitivity can reach 0.01 percent, the DNA amount of a sample can be reduced to aM level, and the detection of low-abundance mutant genes in liquid biopsy of people can be better performed. The present invention has been completed based on this finding. The present invention has been completed based on this finding.

Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.

As used herein, the terms "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …" or "consisting of …".

"transduction," "transfection," "transformation" or terms as used herein refer to the process of delivering an exogenous polynucleotide into a host cell, transcription and translation to produce a polypeptide product, including the introduction of the exogenous polynucleotide into the host cell (e.g., E.coli) using a plasmid molecule.

"Gene expression" or "expression" refers to the process of transcription, translation and post-translational modification of a gene to produce the RNA or protein product of the gene.

"Polynucleotide" refers to a polymeric form of nucleotides of any length, including Deoxynucleotides (DNA), Ribonucleotides (RNA), hybrid sequences thereof, and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to interchangeable single-and double-stranded molecules. Unless otherwise indicated, a polynucleotide in any of the embodiments described herein includes a double-stranded form and two complementary single strands that are known or predictable to make up the double-stranded form.

Conservative amino acid substitutions are known in the art. In some embodiments, the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine. In addition, the invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.

Those skilled in the art will readily understand the meaning of all parameters, dimensions, materials and configurations described herein. The actual parameters, dimensions, materials and/or configurations will depend upon the specific application for which the invention is being used. It will be understood by those skilled in the art that the embodiments or claims are given by way of example only and that within the scope of equivalents or claims, the embodiments of the invention may be covered without limitation to the specifically described and claimed scope.

All definitions, as defined and used herein, should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.

All references, patents, and patent applications cited herein are hereby incorporated by reference with respect to the subject matter to which they are cited, and in some cases may contain the entire document.

It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these embodiments.

Ago enzyme

The Argonaute protein belongs to the PIWI (P element-induced wimpy testis) protein superfamily, is defined by the existence of the PIWI structural domain, is widely existed in all fields of life, and can be combined with siDNA or siRNA guide strand to specifically silence or cut a complementary nucleic acid target strand. Research shows that Ago plays an important role in immune defense and metabolic regulation of organism cells and may have application potential of artificial gene editing, so that function research aiming at Ago protein becomes a new concern in biological research.

Ago proteins were originally found in eukaryotes and are key players of the RNA interference (RNAi) pathway. The eukaryotic Argonaute protein (eAgos) is used as the core of a multi-protein RNA-induced silencing complex (RISC), can be combined with an siRNA molecule to be used as a guide chain, cuts complementary target RNA, and directly silences the translation of the target RNA; or indirectly silence the target RNA by binding with the target RNA and recruiting other silencing factors to promote the degradation of the target RNA. Thus, eAgos can regulate gene expression post-transcriptionally, protect its host from invading RNA viruses, and preserve genome integrity by reducing transposon mobility.

The Argonaute protein is also present in prokaryotes. Structural and biochemical studies of several prokaryotic ago (pagos) proteins (mainly from thermophilic bacteria and archaea) have shown that they can exert endonuclease action in vitro and host defense action in vivo. pAgos can bind to siDNA guide strands to specifically cleave and guide strand-complementary paired DNA target strands. By 2018, pAgos which are mainly derived from a high-temperature host and are mostly used for gene detection have been reported. The activity is very low under normal temperature conditions, and the gene editing tool cannot be used. In 2019, pAgos from normal-temperature hosts are reported in succession, which can exert DNA-guided DNA shearing activity under normal-temperature conditions and can shear plasmids with low GC content.

As used herein, the terms "programmable endonuclease", "nuclease", "Thermococcus eurythermalis", and "TeAgo enzyme" are used interchangeably to refer to an enzyme as described in the first aspect of the invention.

The wild-type TeAgo enzyme has the amino acid sequence shown as NCBI sequence No. WP _ 050002102.1.

The TeAgo enzyme of the invention may also comprise mutated forms thereof which retain functional activity. The mutant form may contain an amino acid sequence obtained by substitution, deletion, alteration or insertion of one or more amino acid residues based on the sequence shown as NCBI sequence No. WP _050002102.1, or addition of 1 to 10 amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues) at its N-terminus or C-terminus; and the obtained amino acid sequence has a sequence identity of more than or equal to 85% (preferably more than or equal to 90%, more preferably more than or equal to 95%, such as more than or equal to 96%, more than or equal to 97%, more than or equal to 98% or more than or equal to 99%) with the sequence shown as NCBI sequence No. WP _ 050002102.1; and the obtained amino acid sequence has the same or similar functions with the wild TeAgo enzyme.

"A-STAR" detection technique

Core of the inventionThe method is characterized in that a novel nucleic acid cutting tool enzyme TeAgo with single-point nucleic acid recognition specificity and high-temperature stability is developed, and a PCR reaction is coupled to realize the process of cutting-amplifying at the same time, so that' A-STARAgo-mediated Specific Target detection) "technique, principle details are as follows: in the high-temperature denaturation step of each cycle of PCR, dsDNA is denatured and melted into ssDNA, and at the temperature, TeAgo cuts ssDNA of a pair of melted wild type genes under the guidance of a pair of specially designed gDNA respectively, namely, the process can specifically cut the wild type genes and retain mutant type genes; in the subsequent PCR annealing step, the designed primers are positioned at least 20nt upstream and downstream of the SNV site of the target nucleic acid, so that the primers can non-selectively bind to a wild-type gene and a mutant gene; in the subsequent PCR extension step, the wild-type gene is cut at the mutation site and cannot be extended as a template, while the mutant gene remains as it is and can be amplified as a template. Because the reaction of coupling the TeAgo high-temperature specific cutting and the PCR amplification can be executed in each cycle of the conventional PCR (20-35 cycles), the cutting-amplification is realized at the same time, thereby efficiently enriching the low-abundance mutant genes.

The technical advantages are that: 1) the high-temperature differential shearing is convenient to operate; 2) the gDNA sequence is matched with a target sequence, and has high specificity; 3) can be designed for any target sequence, without sequence preference; 4) a single enzyme realizes multiple detections on multiple nucleic acid targets; 5) multiple terminal detection techniques may be combined.

Coupled reaction of PCR and shearing

In the present invention, in the coupling reaction of "PCR-cleavage while" using the TeAgo-gDNA complex, the reaction may be carried out under suitable conditions using the corresponding cleaving enzyme and the corresponding amplifying enzyme, as long as the cleaving enzyme and the amplifying enzyme can exert their respective functions under the conditions.

The present study shows that some key factors for enriching mutant dsDNA signals by the coupling reaction mainly include the following aspects:

enriching the initial template concentration in the reaction system: total concentrations (nM. about.fM)) of wild type (wt) and mutant (mutat type, mut): preferably 0.1-100 nM.

② the concentration of the initial TeAgo protein in the enrichment reaction system: preferably 20-100 nM;

③ 94 ℃ of TeAgo-gDNA complex pretreatment time (Pre-processing time (min)): preferably 3-10 minutes;

fourthly, enriching the initial gDNAs concentration in the reaction system: preferably 200-;

fifth, the molar concentration ratio between the TeAgo protein and the gDNAs: preferably 1: 5-1: 20;

sixthly, enriching the cycle number cycle of PCR: preferably 10 to 30;

reaction system for enriching low-abundance target nucleic acid

As used herein, the terms "reaction system for enriching a low abundance target nucleic acid" and "enrichment system of the present invention" are used interchangeably and refer to the reaction system for enriching a low abundance target nucleic acid as described in the second aspect of the present invention.

In the invention, a reaction system for enriching low-abundance target nucleic acid is provided, which is based on the counting principle of the coupling reaction of PCR-shearing, and is used for simultaneously carrying out Polymerase Chain Reaction (PCR) and nucleic acid cutting reaction on a nucleic acid sample so as to obtain an amplification-cutting reaction product.

In a specific embodiment, the nucleic acid sample comprises a first nucleic acid and a second nucleic acid in an enrichment system, wherein the first nucleic acid is the target nucleic acid and the second nucleic acid is a non-target nucleic acid; the nucleic acid cleavage reaction is used to specifically cleave non-target nucleic acids, but not the nucleic acid of interest.

In the enrichment system of the present invention, the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) a nucleic acid cleavage system of the present invention based on the programmable endonuclease Argonaute (ago).

In the enrichment system of the present invention, the concentration of the low-abundance target nucleic acid before enrichment is 0.5-5nM, preferably 0.8-2nM, and more preferably 1 nM.

In addition, the reagents required for performing the PCR reaction also include amplification primer pairs for the target nucleic acid. Preferably, the concentration of each primer in the amplification primer pair of the target nucleic acid is 100-300nM, preferably 150-250nM, and more preferably 200 nM.

In the enrichment system of the present invention, the gDNA comprises forward gDNA and reverse gDNA; wherein, the forward gDNA is gDNA having the same sequence segment as the target nucleic acid, and the reverse gDNA is gDNA having a reverse complementary sequence segment with the target nucleic acid.

In a preferred embodiment, the reaction system further comprises a divalent metal ion. Preferably, the divalent metal ion is Mn²⁺. And the concentration of the divalent metal ion is 50mM-2M, preferably 100mM-1M, more preferably 0.5 mM.

In the process of enriching the target nucleic acid, preferably, the reaction temperature (reaction program) of the reaction system is: 94 ℃ for 5 min; the number of cycles is 10-30(94 ℃, 30 s; 52 ℃, 30 s; 72 ℃, 20 s); 72 ℃ for 1 min.

The invention relates to a method for enriching and detecting low-abundance target nucleic acid

As used herein, the terms "enrichment method of the present invention", "method for enriching a low-abundance target nucleic acid", and "method for enriching a nucleic acid of the present invention" are used interchangeably and all refer to the method for enriching a low-abundance target nucleic acid according to the third aspect of the present invention.

The invention provides a method for enriching low-abundance target nucleic acid, which comprises the following steps: (a) providing a nucleic acid sample, wherein the nucleic acid sample comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is the target nucleic acid, the second nucleic acid is a non-target nucleic acid, and the abundance of the target nucleic acid in the nucleic acid sample is F1 a; (b) performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction in an amplification-cleavage reaction system by taking the nucleic acid in the nucleic acid sample as a template, thereby obtaining an amplification-cleavage reaction product; wherein the nucleic acid cleavage reaction is used to specifically cleave non-target nucleic acids but not the nucleic acid of interest; and, the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) the nucleic acid cleavage system of the present invention; wherein the abundance of said target nucleic acid in said amplification-cleavage reaction product is F1 b; wherein the ratio of F1b/F1a is not less than 10.

In a preferred embodiment, the target nucleic acid and the non-target nucleic acid differ by only one base.

Preferably, the ratio of F1b/F1a is not less than 10 when F1a is not less than 1%, the ratio of F1b/F1a is not less than 100 when F1a is not less than 0.5%, and the ratio of F1b/F1a is not less than 200 when F1a is not more than 0.1%.

As used herein, the terms "detection method of the present invention" and "method for detecting a low-abundance target nucleic acid" are used interchangeably and refer to a method for detecting an enriched low-abundance target nucleic acid based on the method for enriching a low-abundance target nucleic acid according to the third aspect of the present invention.

The invention provides a method for detecting low-abundance target nucleic acid, which is based on the steps of the method for enriching the low-abundance target nucleic acid and further comprises the following steps: (c) detecting the amplification-cleavage reaction product, thereby determining the presence, absence and/or amount of the target nucleic acid.

The detecting in step (c) comprises quantitative detection, qualitative detection, or a combination thereof.

Preferably, said quantitative detection is selected from the group consisting of: TaqMan fluorescent quantitative PCR, Sanger sequencing, q-PCR, ddPCR, chemiluminescence method, high-resolution melting curve method, NGS and the like; more preferably, the fluorescent quantitative PCR is selected from TaqMan fluorescent quantitative PCR and Sanger sequencing.

The detection methods described herein can be non-diagnostic and non-therapeutic.

In practical applications, the nucleic acid sample comprises nucleic acids from a test sample selected from the group consisting of: blood, cells, serum, saliva, body fluids, plasma, urine, prostatic fluid, bronchial lavage, cerebrospinal fluid, gastric fluid, bile, lymph fluid, peritoneal fluid, stool, and the like, or combinations thereof.

In a specific embodiment of the invention, the low-abundance target nucleic acid can be an EGFR E746-A750 mutant sequence fragment or an EGFR L858R mutant sequence fragment.

Reagent kit

Based on the method for enriching and detecting low-abundance target nucleic acid, the invention further provides a kit for detecting target nucleic acid molecules, which comprises: (i) the invention enriches the reaction system of the low-abundance target nucleic acid or the reagent used for preparing the reaction system; (ii) a detection reagent for detecting a low-abundance target nucleic acid; and (ii) instructions for use which describe the detection method of the invention.

In a specific embodiment, the kit comprises: (a) a first container and a guide DNA located in the first container; (b) a second container and a programmable endonuclease, argonaute (ago), located in the second container; and (c) a third container and a reagent for a nucleic acid amplification reaction in the third container.

Preferably, the kit further comprises: (d) a fourth container and a detection reagent of low-abundance target nucleic acid positioned in the fourth container. The detection reagent for the low-abundance target nucleic acid comprises: primers, probes, and the like. In one embodiment, the reagent for detecting a low-abundance target nucleic acid comprises: primers and probes required for TaqMan fluorescent quantitative PCR, or reagents required for Sanger sequencing.

Preferably, the kit further comprises: (e) a fifth container and a divalent metal ion located in the fifth container. Preferably, the kit further comprises: (f) a sixth container and a buffer solution in the sixth container.

In various embodiments of the present invention, the containers may be the same or different containers.

The main advantages of the invention include:

1) an Ago which can withstand an ultra-high temperature and has excellent stability is provided.

2) Provides an Ago which can better distinguish the mismatch between target and gDNA.

3) Provides a nucleic acid tool enzyme which can be applied to gene detection and successfully and efficiently enrich low-abundance mutant DNA.

4) Provides a nucleic acid tool enzyme with gene manipulation potential.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1: obtaining of the sequence of the TeAgo Gene

In a database, similarity retrieval is carried out on the known amino acid sequence of Pfago, partial amino acid sequences with high sequence consistency are selected, MEGA software is adopted for analysis, a homologous evolutionary tree is constructed, and TeAgo is selected as a candidate enzyme, wherein the sequence similarity of the TeAgo and the Pfago which is characterized is 33.02%. The amino acid sequence of TeAgo (WP _050002102.1) and the corresponding gene sequence encoding the protein (NZ _ CP008887.1) were obtained. The gene sequence is optimized and synthesized by a codon and then cloned to a pET28a expression vector.

Example 2: heterologous expression and purification of TeAgo protein

The above-mentioned prokaryotic expression plasmid of TeAgo-pET28a was introduced into E.col I BL21(DE3) to obtain a prokaryotic expression strain of TeAgo-pET28a/E.col I BL21(DE 3). Coli BL21(DE3) containing recombinant plasmid Teago-pET28a was inoculated in LB medium containing 50. mu.g/mL kanamycin and shake-cultured at 37 ℃ and 220rpm to OD₆₀₀Adding IPTG with final concentration of 0.4-0.6mM into the mixture to reach 0.6-0.8 ℃, continuously culturing for 16-20h by using a shaker at 200rpm at 18 ℃, and inducing the expression of the TeAgo protein. The cells were collected by centrifugation and resuspended in a buffer (20mM Tris-HCl, pH8.0 or so)Right, 500mM NaCl) was resuspended, and then the cells were crushed under high pressure and centrifuged to obtain a supernatant. And (3) carrying out affinity purification on the protein by using a Ni-NTA column, and carrying out ultrafiltration concentration, desalination and the like on the eluent to obtain the purified protein. The purified protein was stored in a buffer containing 20mM Tris-HCl and the protein was assayed by BCA kit, the assay procedure was performed according to the protocol. And preparing a standard solution by taking BSA as a standard substance, drawing a standard curve, calculating the concentration of the purified target protein according to the standard solution, and storing the protein in a refrigerator at the temperature of minus 80 ℃ for later use. The TeAgo protein was analyzed by SDS-PAGE electrophoresis.

The results are shown in FIG. 2. The results showed that a clear band was obtained at 88.1kDa, indicating that the protein of interest, TeAgo, was purified.

Example 3: teago shear activity assay

Designing a 45nt single-stranded DNA with fluorescent modification, an RNA target nucleic acid and four complementary 16nt DNAs and RNA guide chains, and sending the DNA and RNA guide chains to a company for synthesis.

DNA target nucleic acid sequence (SEQ ID NO: 21):

5’-FAM-CGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGG-3’

RNA target nucleic acid sequence (SEQ ID NO: 22):

5’-FAM-CGCAGCAUGUCAAGAUCACAGAUUUUGGGCUGGCCAAACUGCUGG-3’

gDNA sequence (SEQ ID NO: 23):

5’-HO/P-TAGTTTGGCCAGCCCA-3’

gRNA sequence (SEQ ID NO: 24):

5’-HO/P-UAGUUUGGCCAGCCCA-3’

reaction buffer (containing 15mM Tris-HCl pH8.0, 250mM NaCl) was prepared, and MnCl was added to the reaction buffer to a final concentration of 0.5mM₂400nM Teago, 2. mu.M synthesized gDNA or gRNA and 0.8. mu.M 5' fluorescence modified sequence complementary single-stranded DNA or RNA target nucleic acid, reacting at 95 ℃ for 15min, taking 6-10. mu.L of sample after reaction, adding sample buffer (containing 95% (deionized) formamide, 0.5mmol/L EDTA, 0.025% bromophenol blue, 0.025% dimethyl phenol blue) according to the ratio of 1:1Bluish) under 16% nucleic acid Urea-PAGE.

The results are shown in FIG. 3. The results show that TeAgo can cleave ssDNA and ssRNA, respectively, using 5 '-OH and 5' -P modified gDNA.

Example 4: analysis of TeAgo catalytic Properties

Respectively detecting enzyme activity of TeAgo at different temperatures (60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C, 100 deg.C), and adding MnCl with final concentration of 0.5mM into reaction buffer solution₂The target nucleic acid of TeAgo with the final concentration of 400nM, gDNA synthesized by 2 muM and complementary single-stranded DNA with the sequence of 0.8 muM 60nt were reacted for 15min at different temperatures, and the reaction product was electrophoretically detected under 16% nucleic acid Urea-PAGE.

The results are shown in FIG. 4. The results show that TeAgo has high-efficiency shearing activity in the range of 90-99.9 ℃.

The thermal stability of TeAgo was determined at temperatures of 90 ℃ and 95 ℃ respectively, with unchanged conditions: firstly, adding MnCl into a reaction buffer solution₂TeAgo and gDNA, respectively at 90 deg.C and 95 deg.C for 0, 5min, 10min, 15min, 20min, 25min and 30 min. The reaction product was examined under the same conditions.

The results are shown in FIG. 5. The results show that TeAgo has good thermal stability at 90 ℃ and 95 ℃, and the shearing activity is not reduced after incubation for 30 min.

The concentrations of the TeAgo, the guide strand and the target nucleic acid are unchanged, and CoCl with the final concentration of 0.5mM is respectively added into a reaction system₂、CuCl₂、MgCl₂、MnCl₂、ZnCl₂、CaCl₂The solution is reacted for 15min at the reaction temperature of 90 ℃, and electrophoresis is carried out under 16 percent nucleic acid Urea-PAGE to detect the influence of metal ions on the activity of the enzyme.

The results are shown in FIG. 6A. The results show that TeAgo utilizes only MnCl₂As the active metal ion.

The reaction system and conditions are unchanged, and different MnCl is added₂Concentration: 25 μ M, 50 μ M, 100 μ M, 250 μ M, 500 μ M, 1000 μ M, 2000 μ M, and the Teago-optimum MnCl was determined under 5' phosphorylation guide chain mediation₂And (4) concentration.

The results are shown in FIG. 6B. The results show that 25-2000mM MnCl₂The TeAgo can keep higher activity.

11-30nt 5' phosphorylated gDNAs are designed respectively, and the influence of gDNAs with different lengths on the activity of the TeAgo enzyme is researched. MnCl was added to the reaction buffer to a final concentration of 0.5mM₂The target nucleic acids of the target DNA of TeAgo with the final concentration of 400nM, gDNA with different lengths synthesized by 2 muM and complementary single-stranded DNA with the sequence of 0.8 muM and 60nt are respectively reacted for 15min at 95 ℃, and the reaction products are subjected to electrophoretic detection under 16% nucleic acid Urea-PAGE.

The results are shown in FIG. 7. The results show that TeAgo can use 15-21nt gDNA to carry out conventional shearing between 10-11 sites; however, when the gDNA length was increased to 30nt, TeAgo appeared in an unconventional manner of cleavage, resulting in the formation of multiple strands of product.

The reaction buffer components were adjusted, reaction buffers were prepared to a final concentration of 15mM Tris-HCl pH8.0 and different concentrations of NaCl (20mM, 50mM, 100mM, 250mM, 500mM, 1000mM, 2000mM, 3000mM, 4000mM, 5000mM) respectively, the reaction system was not changed, the reaction was carried out at 95 ℃ for 15min, and the detection was carried out by electrophoresis on 16% nucleic acid Urea-PAGE.

The results are shown in FIG. 8. The result shows that when the concentration of NaCl in the reaction buffer solution is 20-500mM, the TeAgo can keep higher activity; when NaCl was too high, the shearing activity of TeAgo was inhibited.

The synthesis of phosphorylated gDNA with different modifications at the 5' end was designed: 5 '-P, 5' -OH, 5 '-Biotin, 5' -NH₂5 '-FAM and 5' -SH, adding MnCl without changing a reaction system₂gDNA and target DNA, the cleavage efficiency of TeAgo under 5' different modified guide strand mediation was determined. The reaction was carried out at 95 ℃ for 15min, and the detection was carried out by electrophoresis on 16% nucleic acid Urea-PAGE.

The results are shown in FIG. 9. The results show that TeAgo has a significant preference for 5 'end modification of gDNA, using only 5' -P modified gDNA.

Designing 60-90nt wild type gene and mutant gene with single base mutation, and a series of gDNAs with single point mismatch with target at different sites (MP2-15), adding MnCl₂And gDNA and target DNA mismatched at different sites, and determining the differential shearing effect of the TeAgo. The reaction was carried out at 95 ℃ for 15min, and the detection was carried out by electrophoresis on 16% nucleic acid Urea-PAGE.

The principle and results of primer design are shown in FIG. 10. The results show that TeAgo has low tolerance to single base mutation, and single site mismatching can cause remarkable reduction of the shearing activity.

Designing 60-90nt wild type gene and single base mutant gene, and a series of gDNAs with continuous double-point mismatch at 10-11 site with target, adding MnCl₂And gDNA and target DNA mismatched at different sites, and determining the differential shearing effect of the TeAgo. The reaction was carried out at 95 ℃ for 15min, and the detection was carried out by electrophoresis on 16% nucleic acid Urea-PAGE.

The principle and results of primer design are shown in FIG. 11. The result shows that the 10-11 bit continuous double-dot staggered pairing TeAgo activity has larger influence; however, for the two single strands of EGFR L858R, there still exists gDNA of which WT and Mut genes can be distinguished by TeAgo, and candidate gDNAs are provided for subsequent enrichment of the Mut gene.

Example 5: enrichment of mutant dsDNA by the TeAgo-gDNA Complex

Specific amplification primers, gDNAs and detection probes are designed aiming at the sequence characteristics of the EGFR del E746-A750 gene segment. The specific sequences are shown in tables 1 and 2.

TABLE 1

TABLE 2

Wild and mutant templates were amplified by PCR using EGFR del E746-A750 wild and mutant fragments as substrates, respectively. After the PCR product was purified and recovered, the formulated samples were quantified using the Pikogreen dsDNA quantification kit (hypersensitivity) (Qubit 3.0 compatible) sold by Liji Bio Inc. The template was configured as 1nM 5.0%, 1.0%, 0.01% mut EGFR del E746-A750 samples.

The enrichment PCR reaction program of TeAgo on 1nM EGFR del E746-A750 mutant gene includes: 94 ℃ for 5 min; the number of cycles is 10-30(94 ℃, 30 s; 52 ℃, 30 s; 72 ℃, 20 s); 72 ℃ for 1 min.

The reaction system comprises 2 XPCR Taq Master Mix, 40-100nM Teago, forward and reverse amplification primers each 200nM, a final concentration of 1nM template, 800-2000nM forward and reverse gDNAs, 0.5mM MnCl₂。

Example 6: detection of wild type and mutant DNA products after enrichment of EGFR del E746-A750 mutant Gene

In this example, the enriched product was quantitatively analyzed by TaqMan fluorescent quantitative PCR. First, a standard curve for detecting EGFR del E746-A750 low abundance mutant DNA substrate by the double TaqMan probe method was determined. 157bp wild type and 142bp mutant EGFR del E746-A750 genes were mixed at a 1:1 ratio and diluted in gradient to 1nM, 100pM, 10pM, 1pM, 100fM, 10fM, ddH₂O, plotted as a standard curve, as shown in FIG. 12.

Taking 20 μ L system as an example, the TaqMan-qPCR detection system conditions are as follows: 2 × Vazyme Mix, wild type probe 0.5 μ M, mutant probe 0.5 μ M, forward and reverse probe primers 0.25 μ M, and diluted template 5 μ L. The TaqMan-qPCR program is as follows: 95 ℃ for 8 min; (95 ℃, 15 s; 60 ℃, 40 s).

After the calibration curve is determined, the enriched product is detected, the template is diluted by 100-fold and 1000-fold before detection, and the procedure and the reaction system are as described above.

The enrichment results were processed as shown in fig. 13. The result shows that TeAgo can perform high-efficiency enrichment approaching 100% on Mut genes with allele mutation frequency (VAF) of 5.0% and 1.0% aiming at EGFR del E746-A750 gene; for Mut genes with VAF of 0.1%, TeAgo can also be enriched by about 60%.

Example 7: enrichment of EGFR L858R mutant dsDNA by Teago-gDNA complex

First, gDNA screening was performed: designing a wild type gene in the range of 60-90nt, a mutant type gene with single base mutation and a series of gDNAs with continuous double-point mismatch with target at 10-11 sites according to the sequence characteristics of two chains of EGFR L858R gene, keeping the reaction system unchanged, adding MnCl₂And gDNA and target DNA mismatched at different sites, and determining the differential shearing effect of the TeAgo. The reaction was carried out at 95 ℃ for 15min, and the detection was carried out by electrophoresis on 16% nucleic acid Urea-PAGE. The results are shown in FIG. 11 (ABCD above).

Specific amplification primers, gDNAs and detection probes are designed aiming at the sequence characteristics of the EGFR L858R gene fragment. See table 3, table 4 sequence specifically.

TABLE 3

TABLE 4

Wild and mutant templates were amplified by PCR using EGFR L858R wild and mutant fragments as substrates, respectively. After the PCR product was purified and recovered, the formulated samples were quantified using the Pikogreen dsDNA quantification kit (hypersensitivity) (Qubit 3.0 compatible) sold by Liji Bio Inc. The templates were configured as 1 nM5.0%, 1.0% mut EGFR L858R samples.

The enrichment PCR reaction program of TeAgo on 1nM EGFR L858R mutant gene includes: 94 ℃ for 5 min; the number of cycles is 10-30(94 ℃, 30 s; 52 ℃, 30 s; 72 ℃, 20 s); 72 ℃ for 1 min.

The reaction system comprises 2 XPCR Taq Master Mix, 40-100nM Teago, forward and reverse amplification primers each 200nM, a final concentration of 1nM template, 800-2000nM forward and reverse gDNA, 0.5mM MnCl₂。

Example 8: detection of wild-type and mutant DNA products after enrichment of EGFR L858R mutant Gene

In this example, the enriched product was quantitatively analyzed by TaqMan fluorescent quantitative PCR. First, a standard curve for detecting EGFR L858R low-abundance mutant DNA substrate by a double TaqMan probe method was determined. 148bp wild type and 148bp mutant EGFR L858R genes were mixed at a 1:1 ratio and diluted in gradient to 100pM, 10pM, 1pM, 100fM, 10fM, 1fM, 100aM, ddH₂O, standard curve is shown in FIG. 14. Taking 20 μ L system as an example, the TaqMan-qPCR detection system conditions are as follows: 2 × Vazyme Mix, wild type probe 0.5 μ M, mutant probe 0.5 μ M, forward and reverse probe primers 0.25 μ M, and diluted template 5 μ L. The TaqMan-qPCR program is as follows: 95 ℃ for 8 min; (95 ℃, 15 s; 60 ℃, 40 s).

The enrichment results were processed as shown in figure 15. The result shows that the TeAgo can perform high-efficiency enrichment of nearly 70% on the Mut gene with allele mutation frequency (VAF) of 5.0% aiming at the EGFR L858R gene; for Mut genes with 1% VAF, TeAgo can also be slightly enriched.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai university of transportation

<120> characterization and application of novel high-temperature Argonaute protein

<130> P2021-0608

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<400> 3

ctgtcatagg gactctggat 20

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR-157RV

<400> 4

gcctgaggtt cagagccat 19

<210> 5

<211> 16

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR del E746-FW gDNA

<400> 5

taaggaatta agagaa 16

<210> 6

<211> 15

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR del E746-RV gDNA

<400> 6

ttgcttctct taatt 15

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR (WT) -TaqMan probe

<400> 7

cttctcttaa ttccttgata gcgacgg 27

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR del E746-A750-TaqMan probe

<400> 8

cgctatcaaa acatctccga aagcc 25

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR-Forward primer

<400> 9

ccagaaggtg agaaagtta 19

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR-reverse primer

<400> 10

tcgaggattt ccttgttg 18

<210> 11

<211> 148

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR L858R WT(148bp)

<400> 11

ctacttggag gaccgtcgct tggtgcaccg cgacctggca gccaggaacg tactggtgaa 60

aacaccgcag catgtcaaga tcacagattt tgggctggcc aaactgctgg gtgcggaaga 120

gaaagaatac catgcagaag gaggcaaa 148

<210> 12

<211> 148

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR L858R Mut(148bp)

<400> 12

ctacttggag gaccgtcgct tggtgcaccg cgacctggca gccaggaacg tactggtgaa 60

aacaccgcag catgtcaaga tcacagattt tgggcgggcc aaactgctgg gtgcggaaga 120

gaaagaatac catgcagaag gaggcaaa 148

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR-157FW

<400> 13

ctgtcatagg gactctggat 20

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR-157RV

<400> 14

gcctgaggtt cagagccat 19

<210> 15

<211> 16

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> L858R RV-10T(di-P)

<400> 15

tagtttggct agccca 16

<210> 16

<211> 16

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> L858R FW-10T(di-P)

<400> 16

tattttgggg tggcca 16

<210> 17

<211> 15

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR L858R Probe-WT

<400> 17

agtttggcca gccca 15

<210> 18

<211> 16

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR L858R Probe-Mut

<400> 18

agtttggccc gcccaa 16

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR L858R 148-PF

<400> 19

ccgcagcatg tcaagatcac 20

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> EGFR L858R 148-PR

<400> 20

tccttctgca tggtattctt tctct 25

<210> 21

<211> 45

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> ssDNA

<400> 21

cgcagcatgt caagatcaca gattttgggc tggccaaact gctgg 45

<210> 22

<211> 45

<212> RNA

<213> Artificial sequence (artificial sequence)

<220>

<223> ssRNA

<400> 22

cgcagcaugu caagaucaca gauuuugggc uggccaaacu gcugg 45

<210> 23

<211> 16

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gDNA

<400> 23

tagtttggcc agccca 16

<210> 24

<211> 16

<212> RNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA

<400> 24

uaguuuggcc agccca 16

Claims

1. A nucleic acid cleavage system, comprising:

(a) guide dna (gdna);

(b) the programmable endonuclease, argonaute (ago); and

2. The nucleic acid cleavage system of claim 1, wherein the programmable endonuclease Argonaute is Thermococcus eurytherma (Thermococcus eurytherma) and the programmable endonuclease Argonaute is a programmable endonuclease TeAgo.

3. The nucleic acid cleaving system of claim 1, wherein the guide DNA is a single stranded DNA molecule phosphorylated at the 5' end.

4. The nucleic acid cleavage system of claim 1, wherein the guide DNA has a length of 5 to 30nt, preferably 15 to 21nt, and most preferably 16 to 18 nt.

5. A reaction system for enriching low-abundance target nucleic acid, wherein the reaction system is used for simultaneously performing Polymerase Chain Reaction (PCR) and nucleic acid cleavage reaction on a nucleic acid sample, thereby obtaining an amplification-cleavage reaction product;

the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) the nucleic acid cleavage system of claim 1.

6. The reaction system of claim 5, wherein the gDNA comprises forward gDNA and reverse gDNA;

7. A method for enriching a low abundance target nucleic acid, comprising the steps of:

and, the amplification-cleavage reaction system comprises (i) reagents required for performing a PCR reaction and (ii) the nucleic acid cleavage system of claim 1;

wherein the ratio of F1b/F1a is not less than 10.

8. The method of claim 7, wherein the target nucleic acid and the non-target nucleic acid differ by only one base.

9. A kit for detecting a target nucleic acid molecule, the kit comprising:

(i) the reaction system for enriching low-abundance target nucleic acid or the reagent for preparing the reaction system according to claim 5;

(ii) a detection reagent for detecting a low-abundance target nucleic acid; and

(ii) instructions for use, the instructions describing the method of claim 7.

10. The application of the programmable endonuclease Argonaute is characterized in that the programmable endonuclease Argonaute is used for preparing a reagent or a kit for detecting a target molecule or a reagent or a kit for detecting low-abundance target nucleic acid.