WO2022228351A1 - Representation and application of novel high temperature argonaute protein tpsago - Google Patents

Abstract

Provided are a representation and application of a novel high temperature Argonaute protein. Specifically, provided is a nucleic acid cleaving system, which is characterized in that the nucleic acid cleaving system comprises: (a) a guide DNA (gDNA); (b) a programmable endonuclease Argonaute (Ago); and (c) optional reporter nucleic acid, wherein if the reporter nucleic acid is cleaved, the cleavage can be detected. In addition, further provided are a system and a method for enriching and detecting low-abundance target nucleic acids of the programmable endonuclease TpsAgo. The provided detection system and method can specifically target low-abundance nucleic acids for effective enrichment and efficient detection, and the programmable endonuclease TpsAgo has a strong potential for gene manipulation.

Description

Characterization and application of a novel high temperature Argonaute protein TpsAgo technical field

The invention belongs to the field of biotechnology, and in particular relates to the characterization and application of a novel high-temperature Argonaute protein TpsAgo.

Background technique

The Argonaute (Ago) protein was first mentioned in a study describing mutants of Arabidopsis thaliana. Ago proteins are key players in the eukaryotic RNA interference (RNAi) pathway, which can regulate gene expression post-transcriptionally, thereby defending against host-invading RNA viruses and protecting genome integrity. The reported Ago proteins are mainly divided into two categories: eukaryotic Ago (eAgo) and prokaryotic Ago (pAgo).

Prokaryotic Ago can bind to single-stranded guide DNA or RNA, and catalyze the cleavage of target DNA or RNA that is complementary to the guide. Different from the CRISPR/Cas system, prokaryotic Ago does not require a PAM sequence when cutting the target nucleic acid strand. It can bind to the guide DNA or RNA complementary to the target nucleic acid and cut at any complementary position of the target.

High-temperature Ago proteins derived from archaea can exert shearing activity above 70 °C. The high-temperature Agos that have been characterized so far can generally be combined with single-stranded guide DNA or RNA to shear target single-stranded DNA under high temperature conditions, and a few can also shear Cut the target single-stranded RNA.

In recent years, the concept of "liquid biopsy" is emerging. Obtaining tumor gene mutation information has become a trend. Although circulating tumor DNA is a good surrogate sample of tumor tissue, the detection of circulating tumor DNA requires extremely sensitive techniques due to the scarcity of circulating tumor DNA. The current detection technology mainly relies on next-generation sequencing and digital PCR technology, but they all have certain limitations in terms of sensitivity and operating cost.

Therefore, there is an urgent need in the art to develop a method for enrichment and detection of low-abundance mutant DNA with high specificity and sensitivity.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a high-specificity, high-sensitivity low-abundance mutant DNA enrichment and detection method.

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

(a) guide DNA (gDNA);

(b) the programmable endonuclease Argonaute (Ago); and

(c) An optional reporter nucleic acid, wherein the cleavage is detectable if the reporter nucleic acid is cleaved.

In another preferred embodiment, the temperature of the nucleic acid cutting system is 65-90°C, preferably 70-85°C, more preferably 75-85°C.

In another preferred embodiment, the programmable endonuclease Argonaute is selected from the following group: TtAgo, TpsAgo, SeAgo, RsAgo, KpAgo, hAgo1, hAgo2, MpAgo, TpAgo, PfAgo, MjAgo, MfAgo, NgAgo, LrAgo, AaAgo, CbAgo, CpAgo, IbAgo, KmAgo.

In another preferred embodiment, the programmable endonuclease Argonaute is derived from Thermus parvatiensis, and the programmable endonuclease Argonaute is a programmable endonuclease TpsAgo.

In another preferred embodiment, the TpsAgo includes wild-type and mutant TpsAgo.

In another preferred example, the amino acid sequence of the wild-type programmable endonuclease TpsAgo is shown in NCBI sequence number WP_060384876.1.

In another preferred embodiment, the 5' end of the guide DNA has a modification selected from the group consisting of 5'-P, 5'-OH, 5'-Biotin, 5'-NH ₂ C ₆ , 5'- FAM, or 5'-SHC ₆ .

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

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 and the reporter nucleic acid have a reverse complementary fragment.

In another preferred embodiment, the length of the guide DNA is 5-30nt, preferably 10-24nt, more preferably 16-24nt, more preferably 16-21nt.

In another preferred embodiment, the guide DNA is a single-stranded DNA molecule phosphorylated at the 5' end, and its length is 16nt-21nt.

In another preferred embodiment, the guide DNA is a 5'-terminal hydroxylated single-stranded DNA molecule with a length of 16nt-24nt.

In another preferred example, the first nucleotide at the 5' end of the guide DNA is phosphorylated thymine (T), guanine (G), adenine (A) or cytosine (C).

In another preferred embodiment, the reporter nucleic acid is a single-stranded nucleic acid, including single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA).

In another preferred example, when the guide DNA is a single-stranded DNA molecule phosphorylated at the 5' end, the reporter nucleic acid is single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA).

In another preferred embodiment, when the guide DNA is a single-stranded DNA molecule hydroxylated at the 5' end, the reporter nucleic acid is a single-stranded DNA.

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

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

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

In another preferred example, the reporter nucleic acid is a fluorescent reporter nucleic acid, and the fluorescent reporter nucleic acid has a fluorescent group and/or a quenching group.

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

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

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

In another preferred embodiment, the fluorescent reporter nucleic acid is a single-stranded DNA molecule with only a fluorescent group, and the fluorescent group is FAM.

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

In another preferred example, the divalent metal ion is Mn ²⁺ or Co ²⁺ , preferably Mn ²⁺ .

In another preferred embodiment, in the nucleic acid cleavage system, the concentration of divalent metal ions is 80 μM-3 mM, preferably 100 μM-2 mM, more preferably 250 μM-2 mM.

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

In another preferred embodiment, in the buffer, the concentration of NaCl is 0-4800 mM, preferably 40-1600 mM, more preferably 80-800 mM, more preferably 80-400 mM.

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

In another preferred embodiment, when in the reverse complementary region of the gDNA and the reporter nucleic acid, the 2nd, 3rd, 4th, 6th, 8th, 10th, 11th, 13th, 14th or 16th position from the 5' end (more It is preferably the 2nd, 4th, 10th, 11th, 13th or 14th position, more preferably the 2nd, 10th or 13th position) when there is a mismatch with the reporter nucleic acid, it will be significantly reduced. The shear rate of the programmable endonuclease Argonaute.

In another preferred example, the significantly reducing the cleavage rate of the programmable endonuclease Argonaute refers to: under the same reaction conditions, the cleavage rate of the programmable endonuclease Argonaute is reduced by ≥80% , preferably by ≥85%, more preferably by ≥90%.

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

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

In another preferred embodiment, in the nucleic acid cutting system, the concentration of the programmable endonuclease Ago is 10nM-10μM, preferably 50nM-1μM, more preferably 100nM-500nM, and most preferably 200nM .

In another preferred embodiment, in the nucleic acid cutting system, the concentration of the guide DNA is 10 nM-10 μM, preferably 100 nM-3 μM, more preferably 1 μM-2.5 μM, and most preferably 2 μM.

In another preferred embodiment, the reporter nucleic acid is plasmid DNA, and the guide DNA (gDNA) includes forward gDNA and reverse gDNA;

Wherein, the forward gDNA and one strand of the plasmid DNA can form a first reverse complementary region, and the reverse gDNA and the other strand of the plasmid DNA can form a second reverse complementary region.

In another preferred example, in the plasmid DNA, the distance between the first reverse complementary region and the second reverse complementary region is ≤200bp, preferably ≤50bp, more preferably ≤1bp .

In another preferred embodiment, the plasmid DNA is plasmid pUC19.

In another preferred embodiment, the plasmid DNA is in a supercoiled state.

In another preferred example, the plasmid DNA is in a supercoiled state, and the GC content of the plasmid DNA (80bp GC content near the cleavage site) is 20%-35%, preferably 25%- 32%, more preferably 29%.

In another preferred example, the plasmid DNA is in a supercoiled state, and the GC content of the plasmid DNA (the GC content of 80 bp near the cleavage site) is 55%-75%, preferably 62%- 68%, more preferably 65%.

In a second aspect of the present invention, a reaction system for enriching low-abundance target nucleic acid is provided, and the reaction system is used to simultaneously perform Helicase-dependent isothermal DNA amplification on a nucleic acid sample ) and nucleic acid cleavage reaction, thereby obtaining amplification-cleavage reaction product;

Wherein, the nucleic acid sample contains 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 acid, but not cleaving the target nucleic acid;

The amplification-cleavage reaction system contains (i) reagents required for isothermal amplification reaction and (ii) the nucleic acid cleavage system according to the first aspect of the present invention.

In another preferred example, in the reaction system, the concentration of the programmable endonuclease Argonaute (Ago) is 20-200nM, preferably 30-150nM, more preferably 40-100nM, the best The ground is 50 nM.

In another preferred embodiment, the reagents required for the isothermal amplification reaction include tHDA kit (purchased from New England Biolabs).

In another preferred embodiment, the reagents required for the isothermal amplification reaction further include a pair of amplification primers 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 10-300 nM, preferably 50-200 nM, more preferably 100 nM.

In another preferred example, the concentration of the target nucleic acid is 0.1-100 nM, preferably 0.5-50 nM, more preferably 1 nM.

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

Wherein, the forward gDNA refers to the gDNA having the same sequence fragment as the target nucleic acid, and the reverse gDNA refers to the gDNA having the reverse complementary sequence fragment to the target nucleic acid.

In another preferred embodiment, the reaction system further includes: divalent metal ions.

In another preferred example, the divalent metal ion is Mn ²⁺ .

In another preferred example, in the reaction system, the concentration of divalent metal ions is 50 μM-2000 μM, preferably 100 μM-1000 μM, more preferably 250 μM.

In another preferred example, the reaction temperature (reaction procedure) of the reaction system is: 65° C., 1.5-2 h.

In another preferred embodiment, the target nucleic acid is selected from the following group: wild-type EGFR sequence fragment, EGFR L861Q mutant sequence fragment.

In another preferred embodiment, the nucleotide sequence of the wild-type EGFR sequence fragment is shown in SEQ ID NO:5.

In another preferred embodiment, the nucleotide sequence of the EGFR L861Q mutant sequence fragment is shown in SEQ ID NO:6.

In another preferred embodiment, the nucleotide sequences of the forward gDNA and the reverse gDNA are shown in SEQ ID NOs: 7 and 8, respectively.

In another preferred embodiment, the nucleotide sequences of the amplification primer pair of the EGFR L861Q mutant sequence fragment are shown in SEQ ID NOs: 9 and 10, respectively.

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

(a) providing a nucleic acid sample, the nucleic acid sample contains 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 F1a;

(b) using the nucleic acid in the nucleic acid sample as a template, performing isothermal amplification and nucleic acid cleavage reaction in an amplification-cleavage reaction system, thereby obtaining an amplification-cleavage reaction product;

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

And, the amplification-cleavage reaction system contains (i) reagents required for isothermal amplification reaction and (ii) the nucleic acid cleavage system according to the first aspect of the present invention;

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

Wherein, the ratio of F1b/F1a is ≥5 (preferably ≥10).

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

In another preferred example, when 1%≤F1a≤10%, the ratio of F1b/F1a≥10, when 0.1%≤F1a≤0.5%, the ratio of F1b/F1a≥100, when F1a≤0.1%, The ratio of F1b/F1a is ≥200.

In another preferred embodiment, the nucleic acid sample includes a nucleic acid sample that is directly thermally lysed, a nucleic acid sample that is directly lysed by protease, an extracted nucleic acid sample, a nucleic acid sample that has been pre-amplified by PCR, or any nucleic acid-containing sample .

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 nucleotide insertion, deletion, substitution, or a combination thereof.

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

In another preferred embodiment, the abundance of the non-target nucleic acid in the nucleic acid sample is F2a.

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

In another preferred embodiment, the ratio of F2a/F1a is ≥20, preferably ≥50, more preferably ≥100, and most preferably ≥1000 or ≥5000.

In another preferred embodiment, the abundance of the non-target nucleic acid in the amplification-cleavage reaction product is F2b.

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

In another preferred example, the F1b/F2b ≥ 0.5, preferably ≥ 1, more preferably ≥ 2, most preferably ≥ 3 or ≥ 5.

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

In another preferred embodiment, F1a≤0.5%, preferably≤0.2%, more preferably≤0.1%, most preferably≤0.01%.

In another preferred example, F1b≥10%, preferably ≥30%, more preferably ≥50%, and most preferably ≤70%.

In another preferred embodiment, the "reagents required for isothermal amplification reaction" include: helicase and DNA polymerase.

In another preferred example, the "reagents required for isothermal amplification reaction" further include: dNTP, Mg ²⁺ , and isothermal amplification buffer.

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 target region of the target nucleic acid (ie, the first nucleic acid); and the gDNA in the nucleic acid cleavage system also interacts with non- The nucleic acid sequence of the targeting region of the target nucleic acid (ie, the second nucleic acid) forms the second complementary binding region.

In another preferred embodiment, the first complementary binding region contains at least two unmatched base pairs.

In another preferred embodiment, the second complementary binding region contains 0 or 1 unmatched base pair.

In another preferred embodiment, the second complementary binding region contains one unmatched base pair.

In another preferred embodiment, the first complementary binding region contains at least two unmatched base pairs, so that the complex does not cleave the target nucleic acid; and the second complementary binding region contains one unmatched base pair. matched base pairs, 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 the non-target nucleic acid (i.e. the second nucleic acid).

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

In another preferred embodiment, the method further includes:

(c) detecting the amplification-cleavage reaction product to determine the presence and/or amount of the target nucleic acid.

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

In another preferred embodiment, the quantitative detection is selected from the following group: TaqMan fluorescence quantitative PCR, Sanger sequencing, q-PCR, ddPCR, chemiluminescence, high-resolution melting curve method, NGS, etc.; From TaqMan real-time PCR, Sanger sequencing.

In another preferred embodiment, the first nucleic acid includes 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 greater.

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

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

In another preferred embodiment, the nucleic acid sample includes nucleic acid from a sample, wherein the sample is selected from the group consisting of blood, cells, serum, saliva, body fluid, plasma, urine, prostatic fluid, bronchial lavage fluid, cerebrospinal fluid, gastric fluid, bile, lymph fluid, peritoneal fluid, feces, etc., or a combination thereof.

In another preferred embodiment, the low-abundance target nucleic acid is selected from the group consisting of wild-type EGFR sequence fragments and EGFR L861Q mutant sequence fragments.

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

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

(ii) detection reagents for detecting low-abundance target nucleic acids; and

(ii) Instructions for use describing the method according to the third aspect of the invention.

In another preferred embodiment, the kit includes:

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

(b) a second container and a programmable endonuclease Argonaute (Ago) located in the second container; and

(c) The third container and the nucleic acid amplification reaction reagent located in the third container.

In another preferred embodiment, the kit also contains:

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

In another preferred example, the low-abundance target nucleic acid detection reagents include: primers, probes, and the like.

In another preferred embodiment, the low-abundance target nucleic acid detection reagents include: primers and probes required for TaqMan fluorescence quantitative PCR, or reagents required for Sanger sequencing.

In another preferred embodiment, the low-abundance target nucleic acid detection reagent includes the reagent required for Sanger sequencing.

In another preferred embodiment, the kit also contains:

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

In another preferred embodiment, the kit also includes:

(f) A sixth container and buffer located in the sixth container.

In another preferred embodiment, 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 a fifth aspect of the present invention, use of a programmable endonuclease Argonaute is provided, for preparing a reagent or kit for detecting target molecules, or for preparing a reagent or kit for detecting low-abundance target nucleic acid.

In another preferred embodiment, the programmable endonuclease Argonaute is derived from Thermus parvatiensis; or a homologous analog thereof with the same or similar functions.

In another preferred embodiment, the TpsAgo includes wild-type and mutant TpsAgo.

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

(i) the amino acid sequence as set forth in NCBI SEQ ID NO: WP_060384876.1; and

(ii) on the basis of the sequence shown in NCBI sequence number WP_060384876.1, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 10 amino acid residues are added to its N-terminus or C-terminus. base (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues), thereby the obtained amino acid sequence; and the obtained amino acid sequence has the same sequence as shown in NCBI serial number WP_060384876.1 ≥ 85% (preferably ≥ 90%, more preferably ≥ 95%, eg ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99%) sequence identity; and the amino acid sequence obtained possesses the same as (i) same or similar functionality.

The purpose of the present invention is to provide a novel high-temperature nuclease with multiple guide strand and substrate strand cleavage preferences.

In order to achieve the above object, the present invention provides a high-temperature Argonaute-TpsAgo protein, which has high-temperature nuclease activity, and is derived from a strain of thermophilic bacteria Thermus parvatiensis isolated from hot spring water (90-98°C) in northern India RL is the starting strain, which is a high-temperature Ago protein with a small molecular weight reported so far.

In another aspect, the present invention provides a high-temperature Argonaute, a gene of TpsAgo protein, which encodes the above-mentioned high-temperature nuclease TpsAgo protein.

In the present invention, the recombinant plasmid pET28a-TpsAgo is constructed after the gene of TpsAgo protein is excavated and sequence compared. The recombinant plasmid is transformed into Escherichia coli (DE3) to realize the heterologous expression of TpsAgo, and then purified by Ni-NTA gravity column The Ago protein produced by the recombinant strain was obtained.

The molecular weight of the novel high-temperature Ago protein obtained by the invention is about 76kDa, and the enzyme can simultaneously use 5'-phosphorylated gDNA and 5'-hydroxylated gDNA to mediate the cleavage of single-stranded DNA target nucleic acid, and can also use 5'-phosphorylated gDNA and 5'-hydroxylated gDNA to mediate the cleavage of single-stranded DNA target nucleic acid. - Phosphorylated gDNA mediates cleavage of single-stranded RNA target nucleic acids. The optimum reaction temperature range is between 65℃-90℃; Mn ²⁺ and Co ²⁺ can be used as active ions, and 100μM Mn ²⁺ can keep it highly active; the enzyme has a certain tolerance to NaCl concentration, The tolerance range is between 0-3200mM; the enzyme can utilize 16nt-21nt 5'-P gDNA and 16nt-24nt 5'-OH gDNA; the enzyme has a preference for the first base at the 5' end of gDNA, From the shear kinetics results, it can be seen that gDNA whose first hydroxyl group is T and G is preferred; the enzyme can utilize a variety of 5' end modified gDNA, such as 5'-P, 5'-OH, 5'- Biotin, 5'-NH ₂ C ₆ , 5'-FAM, 5-SHC ₆ such as; the enzyme has a low tolerance for single-point mismatch between target and gDNA, which will have a certain degree of SNV gene detection application prospects. The enzyme can cut single-stranded DNA as well as plasmid DNA, and can cut supercoiled DNA into linear DNA at the positions of 29% GC content and 65% GC content of pUC19 plasmid. The enzyme has good discrimination between single-point mismatch and double-point mismatch, and can be combined with isothermal amplification technology to enrich and detect mutant genes such as EGFR L861Q through the coupling reaction of "amplification and shearing".

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows the results of phylogenetic analysis (A) and multiple sequence alignment (B) of TpsAgo.

Figure 2 shows the results of SDS-PAGE electrophoresis analysis of TpsAgo protein. Among them, the lanes from left to right are protein Marker, cell lysis supernatant, cell lysis precipitation, and purified TpsAgo.

Figure 3 shows the results of the assay of the cleavage activity of TpsAgo.

Figure 4 shows the effect of length of 5'-phosphorylated gDNA (A) and 5'-hydroxylated gDNA (B) on TpsAgo cleavage activity.

Figure 5 shows a graph of the results of the optimum temperature range required for the TpsAgo reaction.

Figure 6 shows the results of the effect of divalent metal ion type (A) and concentration (B) on TpsAgo cleavage activity.

Figure 7 shows the results for the NaCl temperature range that TpsAgo can tolerate.

Figure 8 shows the results of the preference of TpsAgo for the first base at the 5' base end of gDNA.

Figure 9 shows the results of TpsAgo's high tolerance for gDNA 5' modification.

Figure 10 shows the results that TpsAgo can differentiate cleavage against single-point mismatches at different sites between gDNA and Target.

Figure 11 shows the results of cleavage of pUC19 plasmids at 29% GC (A, B) and 65% GC (C, D) content positions by TpsAgo.

Among them, OC stands for open-circle plasmid (one strand of plasmid is broken); LIN stands for linearized plasmid (double strand of plasmid is broken); SC stands for supercoiled plasmid.

Figure 12 shows the design principle of forward and reverse gDNAs for TpsAgo enrichment of SNV mutant genes.

Figure 13 shows the enrichment sequencing results of TpsAgo combined with isothermal amplification technology for the EGFR L861Q SNV gene (A means no TpsAgo added, B means 50nM TpsAgo added).

Detailed ways

After extensive and in-depth research and extensive screening, the present inventors have developed for the first time a method for enrichment and detection of low-abundance mutant DNA with high sensitivity, good specificity and high throughput. Specifically, the inventors obtained the nuclease TpsAgo through in vitro expression, purification and separation, and obtained its optimal reaction parameters through a large number of groping experiments, thereby providing a TpsAgo-based method for enriching low-abundance target nucleic acids and corresponding detection methods. The invention has the advantages of non-invasiveness, easy operation, rapidity, etc., and can better detect low-abundance mutant genes in human liquid biopsy. The technology of the invention can be widely used in various fields of molecular diagnosis involving nucleic acid detection, such as tumor liquid Biopsy, infectious diseases such as major infectious and pathogenic infectious diseases (viruses, pathogenic bacteria) detection fields. The present invention has been completed on this basis.

the term

For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined otherwise 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 specific methods and experimental conditions described, as such methods 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, the scope of the invention will be limited only by the appended claims.

Unless otherwise defined, 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, when used in reference to a specifically recited value, the term "about" 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 all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance can, but need not, occur.

As used herein, the terms "containing" or "including (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 the terms used herein refer to the process of delivering an exogenous polynucleotide into a host cell for transcription and translation to produce a polypeptide product, including the use of plasmid molecules to transfer the exogenous polynucleotide to a host cell. The polynucleotide is introduced into a host cell (eg, E. coli).

"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 a gene.

"Polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and the like. Polynucleotides can 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 specified, a polynucleotide in any of the embodiments described herein includes both the double-stranded form and the two complementary single strands known or predicted to make up the double-stranded form.

Conservative amino acid substitutions are known in the art. In some embodiments, potential substituting amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine, and proline; aspartic acid, glutamic acid amino acids; asparagine, glutamine; serine, threonine, lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine . In addition, the present invention also provides non-conservative amino acid substitutions that allow for 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. Actual parameters, dimensions, materials and/or configurations depend on the particular application for which the invention is described. Those skilled in the art will appreciate that the embodiments or claims are given by way of example only, and within the scope of equivalents or claims, the scope that the embodiments of the present invention can cover is not limited to what is specifically described and claimed. scope.

Definitions, and all definitions used herein, should be understood to control over dictionary definitions or definitions in documents incorporated by reference.

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

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 examples.

Ago enzyme

Argonaute protein belongs to the PIWI (P element-induced wimpy testis) protein superfamily, which is defined by the presence of the PIWI domain, widely present in all areas of life, and can bind to siDNA or siRNA guide strand to specifically silence or cut complementary nucleic acids target strand. Studies have shown that Ago plays an important role in the immune defense and metabolic regulation of organisms, and may have the application potential of artificial gene editing. Therefore, the functional study of Ago protein has become a new focus in biological research.

Originally discovered in eukaryotes, Ago proteins are key players in the RNA interference (RNAi) pathway. Eukaryotic Argonaute protein (eAgos), as the core of multi-protein RNA-induced silencing complex (RISC), can bind siRNA molecules as guide strands, cleaves complementary target RNAs, and directly silence the translation of target RNAs; or by binding to target RNAs, Other silencing factors are recruited to promote their degradation, thereby indirectly silencing the target RNA. Thus, eAgos can regulate gene expression post-transcriptionally, protect their hosts from invading RNA viruses, and maintain genome integrity by reducing the mobility of transposons.

Argonaute proteins are also present in prokaryotes. Structural and biochemical studies of some prokaryotic Ago (pAgos) proteins (mainly from thermophilic bacteria and archaea) have shown that they can function as endonucleases in vitro and host defense in vivo. pAgos can bind to the siDNA guide strand to specifically cleave the complementary paired DNA target strand of the guide strand. As of 2018, the reported pAgos are mainly derived from high temperature hosts and are mostly used for genetic testing. The activity is very low at room temperature and cannot be used as a tool for gene editing. Since 2019, some pAgos derived from normal temperature hosts have been reported successively, which can exert DNA-directed DNA shearing activity under normal temperature conditions, and can shear plasmids with low GC content.

As used herein, the terms "programmable endonuclease Thermus parvatiensis", "nuclease Thermus parvatiensis", "TpsAgo enzyme" are used interchangeably and refer to the enzymes described in the first aspect of the invention.

The wild-type TpsAgo enzyme has the amino acid sequence shown in NCBI SEQ ID NO: WP_060384876.1.

The TpsAgo enzymes of the present invention may also comprise mutant forms thereof that retain functional activity. Said mutant form can contain one or more amino acid residues substitution, deletion, change or insertion on the basis of the sequence shown in NCBI sequence number WP_060384876.1, or add 1 to 10 amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues), the amino acid sequence obtained; and the amino acid sequence obtained is the same as NCBI sequence number WP_060384876.1 The sequence shown has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) sequence identity; and the amino acid sequence obtained has Same or similar function as wild-type TpsAgo enzyme.

"Amplification and cleavage" coupling reaction

In the present invention, when the TpsAgo-gDNA complex is used to carry out the coupling reaction of "cleaving while amplification", the reaction can be carried out under appropriate conditions using the corresponding cleavage enzyme and the corresponding amplification enzyme, as long as the conditions are The cleavage enzymes and amplification enzymes can perform their corresponding functions.

The research of the present invention shows that, for enriching the mutant dsDNA signal through the coupling reaction, some key factors mainly include the following aspects:

①The initial template concentration in the enrichment reaction system: wild type (wild type, wt) and mutant type (mutant type, mut) total concentration (nM～fM)): preferably 0.1-100nM.

②Initial TpsAgo protein concentration in the enrichment reaction system: preferably 20-100nM;

③The initial concentration of gDNAs in the enrichment reaction system: preferably 200-2000nM;

④The molar concentration ratio between TpsAgo protein and gDNAs: preferably 1:5～1:20;

A reaction system for enriching low-abundance target nucleic acids

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

In the present invention, a reaction system for enriching low-abundance target nucleic acid is provided. The system is based on the above-mentioned counting principle of the coupling reaction of "amplifying while shearing", and is used for simultaneously performing nucleic acid amplification on a nucleic acid sample. The amplification reaction and the nucleic acid cleavage reaction are carried out to obtain the amplification-cleavage reaction product.

In a specific embodiment, in the enrichment system, the nucleic acid sample contains 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 to specifically cut non-target nucleic acid, but not the target nucleic acid.

In the enrichment system of the present invention, the amplification-cleavage reaction system contains (i) the reagents required for the nucleic acid amplification reaction and (ii) the nucleic acid based on the programmable endonuclease Argonaute (Ago) of the present invention cutting system.

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

In another preferred example, in the reaction system, the concentration of the programmable endonuclease Argonaute (Ago) is 20-200 nM, preferably 30-150 nM, more preferably 40-100 nM.

In another preferred embodiment, the reagents required for the isothermal amplification reaction include One-Step tHDA kit (purchased from NEB Company (New England Biolabs).

In addition, the reagents required for performing the isothermal amplification reaction also include a pair of amplification primers for the target nucleic acid. Preferably, the concentration of each primer in the amplification primer pair of the target nucleic acid is 10-300 nM, preferably 50-200 nM, more preferably 100 nM.

In the enrichment system of the present invention, the gDNA includes forward gDNA and reverse gDNA; wherein, the forward gDNA refers to the gDNA with the same sequence fragment as the target nucleic acid, and the reverse gDNA refers to the gDNA with the target nucleic acid. gDNA of the reverse complement fragment.

In a preferred embodiment, the reaction system further includes divalent metal ions. Preferably, the divalent metal ion is Mn ²⁺ . And the concentration of the divalent metal ion is 50 μM-2000 μM, preferably 100 μM-1000 μM, more preferably 250 μM.

In the process of enriching the target nucleic acid, preferably, the reaction temperature (reaction procedure) of the reaction system is: 65° C., 1.5-2 h.

The method for enriching and detecting low-abundance target nucleic acid of the present invention

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

The present invention provides a method for enriching low-abundance target nucleic acid, comprising the steps of: (a) providing a nucleic acid sample, wherein the nucleic acid sample contains 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 F1a; (b) the nucleic acid in the nucleic acid sample is used as a template, Perform isothermal amplification and nucleic acid cleavage reaction in an amplification-cleavage reaction system to obtain an amplification-cleavage reaction product; wherein the nucleic acid cleavage reaction is used to specifically cut non-target nucleic acid, but not cut the target nucleic acid and, the amplification-cutting reaction system contains (i) reagents required for isothermal amplification reaction and (ii) the nucleic acid cutting system of the present invention; wherein, the target nucleic acid is in the amplification-cutting The abundance in the reaction product is F1b; wherein, the ratio of F1b/F1a is ≥5 (preferably ≥10).

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

Preferably, when 1%≤F1a≤10%, the ratio of F1b/F1a≥10, when 0.1%≤F1a≤0.5%, the ratio of F1b/F1a≥100, when F1a≤0.1%, the ratio of F1b/F1a Ratio≥200.

As used herein, the terms "detection method of the present invention" and "method for detecting low-abundance target nucleic acid" can be used interchangeably, and refer to the method for enriching low-abundance target nucleic acid based on the third aspect of the present invention. A method for the detection of enriched low-abundance target nucleic acids.

The present invention provides a method for detecting low-abundance target nucleic acid, the method is based on the above-mentioned method steps for enriching low-abundance target nucleic acid, further comprising: (c) detecting the amplification-cleavage reaction product , thereby determining the presence and/or quantity of the target nucleic acid.

The detection in the step (c) includes quantitative detection, qualitative detection, or a combination thereof.

Preferably, the quantitative detection is selected from the following group: TaqMan fluorescence quantitative PCR, Sanger sequencing, q-PCR, ddPCR, chemiluminescence, high-resolution melting curve method, NGS, etc.; more preferably, selected from TaqMan fluorescence Quantitative PCR, Sanger sequencing.

The detection methods of the present invention may be non-diagnostic and non-therapeutic.

In practical applications, the nucleic acid sample includes nucleic acid from a sample, wherein the sample is selected from the group consisting of blood, cells, serum, saliva, body fluid, plasma, urine, prostatic fluid, bronchial lavage fluid , cerebrospinal fluid, gastric fluid, bile, lymph fluid, peritoneal fluid and feces, etc. or a combination thereof.

In a specific embodiment of the present invention, the low-abundance target nucleic acid can be an EGFR L861Q mutant sequence fragment.

Reagent test kit

Based on the method for enriching and detecting low-abundance target nucleic acid of the present invention, the present invention further provides a kit for detecting target nucleic acid molecules, comprising: (i) the present invention for enriching the low-abundance target nucleic acid reaction system or reagents for preparing the reaction system; (ii) detection reagents for detecting low-abundance target nucleic acids; and (ii) instructions for use, which describe the detection method of the present 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 located in the second container (Ago); and (c) a third container and a nucleic acid amplification reaction reagent located in the third container.

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

Preferably, the kit further contains: (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 located in the sixth container.

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

The main advantages of the present invention include:

1) The TpsAgo protein of the present invention has a good distinction between single-point or double-point mismatches between target DNA and gDNA. Using this property, it can be applied to the enrichment and enrichment of low-abundance mutant genes. In the detection, the detection of early mutated genes in tumors is realized.

2) TpsAgo can use a variety of 5'-modified gDNA to cleave complementary DNA.

3) TpsAgo can tolerate high concentration of NaCl.

4) TpsAgo can cut a variety of target nucleic acids, including single-stranded DNA, single-stranded RNA, and double-stranded DNA.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacture conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise specified.

Example 1: Acquisition of TpsAgo Gene Sequence

In the database, the known amino acid sequences of PfAgo were searched for similarity, and some amino acid sequences with high sequence consistency were selected, analyzed by MEGA software, and a homologous evolutionary tree was constructed, and TpsAgo was selected as the candidate enzyme. The amino acid sequence of TpsAgo (WP_060384876.1) and the corresponding gene sequence encoding the protein (NZ_CP014142.1) were obtained. After the gene sequence was synthesized by codon optimization, it was cloned into pET28a expression vector.

Example 2: Heterologous expression and purification of TpsAgo protein

The above-mentioned TpsAgo-pET28a prokaryotic expression plasmid was introduced into E.coli BL21(DE3) to obtain a TpsAgo-pET28a/E.coli BL21(DE3) prokaryotic expression strain. The expression strain E.coli BL21(DE3) containing the recombinant plasmid TpsAgo-pET28a was inoculated into LB medium containing 50 μg/mL kanamycin, and incubated at 37°C with a shaker at 220 rpm to an OD ₆₀₀ to 0.6-0.8. IPTG with a final concentration of 0.4-0.6 mM, 18° C., 200 rpm shaker was continued to culture for 16-20 h to induce the expression of TpsAgo protein. The cells were collected by centrifugation, resuspended in a resuspension buffer (containing 20 mM Tris-HCl, about pH 8.0, 1 M NaCl), then disrupted by high pressure, and centrifuged to obtain the supernatant. The protein was affinity purified by Ni-NTA column, and the eluate was concentrated by ultrafiltration, desalted and other steps to obtain the purified protein. The purified protein was stored in a buffer containing 20 mM Tris-HCl, and the protein was assayed by BCA kit, and the assay steps were carried out according to the operating instructions. Using BSA as a standard, prepare a standard solution, draw a standard curve, and calculate the purified target protein concentration based on this, and store the protein in a -80°C refrigerator for later use. TpsAgo protein was analyzed by SDS-PAGE electrophoresis.

The results are shown in Figure 2. The results showed that the target protein TpsAgo had been purified.

Example 3: Alignment of TpsAgo with other known Ago sequences

In this example, TpsAgo was subjected to multiple sequence alignment with some of the characterized Agos.

The results showed that TpsAgo had the smallest number of amino acids and the smallest protein molecular weight. It has been reported in the literature that the targeted cleavage of all catalytically active Ago proteins is mediated by a conserved DEDX (X stands for histidine, aspartate or asparagine) quadruplet. Through sequence alignment, it can be found that there is a DEDD quadruplet in TpsAgo (Figure 1). Therefore, it is further speculated that it may have nuclease catalytic activity, which requires further in vitro identification and characterization.

Example 4: TpsAgo cleavage activity assay

Design 45nt single-stranded DNA with fluorescent modification, RNA target nucleic acid and four complementary 16nt DNA and RNA guide strands, and send them to the company for synthesis.

DNA target nucleic acid sequence (SEQ ID NO: 1):

5’-FAM-CGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGG-3’

RNA target nucleic acid (SEQ ID NO: 2):

5’-FAM-CGCAGCAUGUCAAGAUCACAGAUUUUGGGCUGGCCAAACUGCUGG-3’

gDNA (SEQ ID NO: 3):

5’-HO/P-TAGTTTGGCCAGCCCA-3’

gRNA (SEQ ID NO: 4):

5’-HO/P-UAGUUUGGCCAGCCCA-3’

Prepare the reaction buffer (containing 15mM Tris-HCl pH8.0, 250mM NaCl), add 0.5mM MnCl ₂ , 200nM TpsAgo, 2μM synthetic gDNA or gRNA and 0.8μM 5' fluorescently modified to the reaction buffer Sequence complementary single-stranded DNA or RNA target nucleic acid, react at 80°C for 30min, after the reaction, take 6-10μL of sample, add loading buffer (containing 95% (deionized) formamide, 0.5mmol/ L EDTA, 0.025% bromophenol blue, 0.025% xylene blue), electrophoresis detection was performed under 16% nucleic acid Urea-PAGE.

The results are shown in Figure 3. The results show that TpsAgo can use 5'-P and 5'-OH gDNA to cleave complementary single-stranded DNA, and can also use 5'-P gDNA to cleave complementary single-stranded RNA.

Example 5: Analysis of the catalytic properties of TpsAgo

11-30nt 5'phosphorylated gDNA and 14-24nt 5'hydroxylated gDNA were designed to explore the effect of different lengths of gDNA on TpsAgo enzyme activity. MnCl ₂ with a final concentration of 0.5 mM, TpsAgo with a final concentration of 200 nM, 2 μM of synthesized gDNA of different lengths, and 0.8 μM of 60nt sequence complementary single-stranded DNA target nucleic acid were added to the reaction buffer, respectively, and reacted at 80 °C for 30 min. Electrophoretic detection was performed under 16% nucleic acid Urea-PAGE.

The results are shown in Figure 4. The results show that TpsAgo can use 16-20nt 5'-P gDNA, and can also use 16-22nt 5'-OH gDNA to cut complementary target nucleic acid.

The enzymatic activity of TpsAgo was investigated at different temperatures (50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C). Add MnCl ₂ with a final concentration of 0.5mM, TpsAgo with a final concentration of 200nM, 2μM synthetic gDNA and 0.8μM 60nt sequence complementary single-stranded DNA target nucleic acid, and react at different temperatures for 30min, and the reaction products are displayed on 16% nucleic acid Urea-PAGE electrophoresis detection.

The results are shown in Figure 5. The results show that TpsAgo can use 5'-P gDNA to cleave complementary target nucleic acid in the range of 65℃-90℃.

The concentrations of TpsAgo, guide strand and target nucleic acid remained unchanged. CoCl ₂ , CuCl ₂ , MgCl ₂ , MnCl ₂ , ZnCl ₂ , and CaCl ₂ solutions with a final concentration of 0.5 mM were added to the reaction system. , the effect of metal ions on enzyme activity was detected by electrophoresis under 16% nucleic acid Urea-PAGE. The reaction system and conditions remained unchanged. Different concentrations of MnCl ₂ were added: 25 μM, 50 μM, 100 μM, 250 μM, 500 μM, 1000 μM, and 2000 μM to determine the optimal MnCl ₂ concentration of TpsAgo mediated by the 5' phosphorylated guide strand.

The results are shown in Figure 6. The results show that TpsAgo can utilize Mn ²⁺ and Co ²⁺ as metal ions to mediate single-stranded DNA-guided DNA cleavage. Among them, TpsAgo prefers Mn ²⁺ , and 100μM-2000μM Mn ²⁺ can keep TpsAgo highly active.

Adjust the composition of the reaction buffer to prepare reaction buffers with a final concentration of 15mM Tris-HCl pH8.0 and different concentrations of NaCl (20mM, 40mM, 80mM, 200mM, 400mM, 800mM, 1600mM, 2400mM, 3200mM, 4000mM, 4800mM). , the other reaction systems were unchanged, and the reaction was carried out at 80 °C for 30 min, and electrophoresis was carried out under 16% nucleic acid Urea-PAGE.

The results are shown in Figure 7. The results showed that TpsAgo could exert shearing activity at NaCl concentration of 0-3200 mM.

The 16nt gDNA whose first base at the 5' end of gDNA is A, T, G, and C was designed respectively. The reaction system was unchanged. MnCl ₂ , gDNA and target DNA were added. The preference of the first base at the 5' end of gDNA. The reaction was carried out at 80°C for 15 min, and electrophoresis was performed under 16% nucleic acid Urea-PAGE. The reaction system was unchanged, and the reactions were performed for 0, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, respectively, and the shearing kinetics of the four kinds of gDNA were determined.

The results are shown in Figure 8. The results show that TpsAgo prefers 5'T or 5'G.

Design and synthesize gDNA with different 5' end modifications: 5'-P, 5'-OH, 5'-Biotin, 5'-NH ₂ C ₆ , 5'-FAM, 5'-SHC ₆ , the reaction system is unchanged, add MnCl ₂ , gDNA and target DNA, and the cleavage efficiency of TpsAgo mediated by different 5' modified guide strands was determined. The reaction was carried out at 80°C for 15 min, and electrophoresis was performed under 16% nucleic acid Urea-PAGE.

The results are shown in Figure 9. The results show that TpsAgo can utilize 5'-P, 5'-OH, 5'-Biotin, 5'-NH ₂ C ₆ , 5'-FAM, 5'-SHC ₆ modified gDNA, and prefer 5'-OH and 5'-NH ₂ C ₆ .

Design wild-type nucleic acid sequences in the range of 60-90nt, mutant nucleic acid sequences with single base mutation, and a series of gDNAs with single-point mismatches with the target at different sites (MP2-15), the reaction system is unchanged, add MnCl ₂ , and mismatched gDNA and target DNA at different sites were used to determine the differential cleavage effect of TpsAgo. The reaction was carried out at 80°C for 15 min, and electrophoresis was performed under 16% nucleic acid Urea-PAGE.

The results are shown in Figure 10. The results showed that the cleavage efficiency of TpsAgo was significantly reduced when mismatches occurred at positions 2, 3, 4, 6, 8, 10, 11, 13, 14 and 16 of the gDNA.

The gDNAs of the two strands of the spliced plasmid pUC19 with GC contents of 29% and 65% (within 80 bp upstream and downstream of the cleavage site) were designed and synthesized. The sequences are shown in Figure 11 . Prepare reaction buffer (containing 15mM Tris-HCl pH8.0, 100mM NaCl), add MnCl ₂ at a final concentration of 0.5mM, 750nM TpsAgo, 2.5μM synthetic forward and reverse gDNA and 300ng-600ng pUC19 plasmid to the reaction buffer , and react at 80°C for 2-4h. After the reaction, a certain amount of proteinase K and CaCl ₂ were added to the sample, and the reaction was carried out at 50-55° C. for 1 h. Add 5× loading buffer and perform electrophoresis detection in 1.2% agarose gel.

The results are shown in Figure 11. The results showed that when the GC content near the cleavage site was 29%, TpsAgo could use a pair of gDNA to cut all the supercoiled plasmids into linear plasmids; when the GC content near the cleavage site was 65%, TpsAgo could use a pair of gDNA to cut all the supercoiled plasmids into linear plasmids. Transform supercoiled plasmids into open-circular and linear plasmids for gDNA.

Example 6: TpsAgo combined with isothermal amplification technology to achieve enrichment detection of EGFR single nucleotide variation (SNV) gene

Taking the EGFR L861Q SNV mutant gene as an example, according to the sequence characteristics (see the sequence in Table 1 for details), gDNAs with mismatches with the SNV gene at positions 10-11 were designed, as shown in Figure 12. The two strands of EGFR were used as target DNAs to screen gDNAs that could differentiate between splicing wild-type and SNV genes. The splicing system was the same as above.

The wild and mutant templates were amplified by PCR using EGFR L861Q SNV wild and mutant fragments as substrates, respectively. After the PCR product was purified and recovered, the prepared samples were quantified using the Pikogreen dsDNA quantification kit (supersensitive) (compatible with Qubit 3.0) sold by Liji Bio. Templates were configured as 10 nM (final concentration of enrichment system) 10% mut EGFR L861Q samples.

After screening the gDNA, the EGFR L861Q SNV gene was enriched using the tHDA kit sold by New England Biolabs in combination with TpsAgo. Take a 50 μL reaction system as an example:

The rest of the components were added according to the instructions of the tHDA kit.

Reaction program: 65℃, 1.5-2h

Enriched samples were detected by Sanger sequencing.

The results are shown in Figure 13. The results showed that for the EGFR L858R gene, TpsAgo could slightly enrich the Mut gene with an allelic mutation frequency (VAF) of 10%.

Table 1

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (15)

1. A nucleic acid cutting system, characterized in that the nucleic acid cutting system comprises:

   (a) guide DNA (gDNA);

   (b) the programmable endonuclease Argonaute (Ago); and

   (c) An optional reporter nucleic acid, wherein the cleavage is detectable if the reporter nucleic acid is cleaved.
2. The nucleic acid cutting system according to claim 1, wherein the temperature of the nucleic acid cutting system is 65-90°C, preferably 70-85°C, more preferably 75-85°C.
3. The nucleic acid cutting system of claim 1, wherein the programmable endonuclease Argonaute is derived from Thermus parvatiensis, and the programmable endonuclease Argonaute is a programmable endonuclease Dicer TpsAgo.
4. The nucleic acid cutting system according to claim 1, wherein the nucleic acid cutting system further comprises: (d) divalent metal ions.
5. The nucleic acid cutting system of claim 1, wherein the 5' end of the guide DNA has a modification selected from the group consisting of 5'-P, 5'-OH, 5'-Biotin, 5'- _NH2C6 , 5'-FAM, or 5' _{- SHC6} .
6. The nucleic acid cutting system according to claim 1, wherein the nucleic acid cutting system further comprises: (e) a buffer.
7. The nucleic acid cutting system according to claim 6, wherein, in the buffer, the concentration of NaCl is 0-4800mM, preferably 40-1600mM, more preferably 80-800mM, more preferably 80 -400mM.
8. The nucleic acid cutting system according to claim 1, wherein the reporter nucleic acid is a single-stranded nucleic acid, including single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA).
9. The nucleic acid cutting system of claim 1, wherein the reporter nucleic acid is plasmid DNA, and the guide DNA (gDNA) comprises forward gDNA and reverse gDNA;

   Wherein, the forward gDNA and one strand of the plasmid DNA can form a first reverse complementary region, and the reverse gDNA and the other strand of the plasmid DNA can form a second reverse complementary region.
10. The nucleic acid cutting system of claim 9, wherein the plasmid DNA is in a supercoiled state.
11. A reaction system for enriching low-abundance target nucleic acid, characterized in that the reaction system is used to simultaneously perform Helicase-dependent isothermal DNA amplification and nucleic acid cleavage reaction on a nucleic acid sample, Thereby the amplification-cleavage reaction product is obtained;

    Wherein, the nucleic acid sample contains 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 acid, but not cleaving the target nucleic acid;

    The amplification-cleavage reaction system contains (i) reagents required for isothermal amplification reaction and (ii) the nucleic acid cleavage system according to claim 1 .
12. A method for enriching low-abundance target nucleic acid, comprising the steps of:

    (a) providing a nucleic acid sample, the nucleic acid sample contains 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 F1a;

    (b) using the nucleic acid in the nucleic acid sample as a template, performing isothermal amplification and nucleic acid cleavage reaction in an amplification-cleavage reaction system, thereby obtaining an amplification-cleavage reaction product;

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

    And, the amplification-cleavage reaction system contains (i) reagents required for isothermal amplification reaction and (ii) the nucleic acid cleavage system according to claim 1;

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

    Wherein, the ratio of F1b/F1a is ≥5 (more preferably ≥10).
13. A test kit for detecting target nucleic acid molecules, characterized in that the test kit comprises:

    (i) the enrichment low-abundance target nucleic acid reaction system of claim 11 or a reagent for preparing the reaction system;

    (ii) detection reagents for detecting low-abundance target nucleic acids; and

    (ii) Instructions for use, which instructions describe the method of claim 12.
14. The use of a programmable endonuclease Argonaute is characterized in that it is used for preparing a reagent or kit for detecting target molecules, or for preparing a reagent or kit for detecting low-abundance target nucleic acid.
15. The use according to claim 14, wherein the programmable endonuclease Argonaute has an amino acid sequence selected from the group consisting of:

    (i) the amino acid sequence as set forth in NCBI SEQ ID NO: WP_060384876.1; and

    (ii) on the basis of the sequence shown in NCBI sequence number WP_060384876.1, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 10 amino acid residues are added to its N-terminus or C-terminus. base (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues), thereby the obtained amino acid sequence; and the obtained amino acid sequence has the same sequence as shown in NCBI serial number WP_060384876.1 ≥ 85% (preferably ≥ 90%, more preferably ≥ 95%, eg ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99%) sequence identity; and the amino acid sequence obtained possesses the same as (i) same or similar functionality.

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