WO2022222920A1

WO2022222920A1 - Characterization and application of novel high-temperature argonaute protein

Info

Publication number: WO2022222920A1
Application number: PCT/CN2022/087651
Authority: WO
Inventors: 冯雁; 孙莹璎; 郭翔; 陆慧; 李忠磊
Original assignee: 上海交通大学
Priority date: 2021-04-20
Filing date: 2022-04-19
Publication date: 2022-10-27
Also published as: CN113106087A; CN113106087B

Abstract

Provided is a nucleic acid cleavage system, comprising a guide DNA, a programmable endonuclease Argonaute, and an optional reporter nucleic acid, the cleavage of which can be detected. Also provided is a method for enriching and detecting a low abundance target nucleic acid, comprising: amplifying the target nucleic acid by means of PCR and specifically cleaving a non-target nucleic acid by the programmable endonuclease Argonaute. The programmable endonuclease Argonaute is TeAgo from Thermococcus eurythermalis.

Description

Characterization and application of a novel high temperature Argonaute protein

technical field

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

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 cleaving the target nucleic acid strand. It can combine with a guide (DNA or RNA) complementary to the target nucleic acid to cleavage at any 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.

Ago protein has also been used in the field of genetic testing in recent years. Due to its selective splicing of wild-type genes and mutant genes, it can be used for the enrichment of low-abundance mutant DNA in tumor-related genes. Reported TtAgo and PfAgo. Among them, using a new nucleic acid cutting tool enzyme PfAgo, coupled with PCR reaction to realize the process of cutting-while-amplification, and the established "A-STAR (Ago-mediated Specific Target detection)" technology, is a highly specific and enrichment technology that can be combined with multi-terminal detection technology. Due to the limitations of current clinical detection methods for rare mutations, the research on new detection technologies and new nucleic acid tool enzymes has increasingly become 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 low-abundance DNA enrichment and detection method.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a more efficient low-abundance 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 said cleavage is detectable if said reporter nucleic acid is cleaved.

In another preferred embodiment, the temperature of the nucleic acid cutting system is 80-99.9°C, preferably 90-99.9°C, more preferably 94-96°C, more preferably 95°C.

In another preferred embodiment, the programmable endonuclease Argonaute is derived from prokaryotes Thermococcus eurythermalis, Methanocaldococcus fervens, Methanocaldococcus jannaschii, Pyrococcus furious, Marinitoga piezophila, Aquifex aeolicus, Clostridium butyricum, Clostridium perfringens, Thermus thermophilus, Natronobacterium gregoryi, Intestinibacter bartlettii, Kurthia masssiliensis, Synechococcus elongatus.

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

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

In another preferred example, the amino acid sequence of the wild-type programmable endonuclease TeAgo is shown in NCBI sequence number WP_050002102.1.

In another preferred example, the guide DNA is phosphorylated at the 5' end, hydroxylated at the 5' end, with a Biotin group at the 5' end, with an NH ₂ C ₆ group at the 5' end, and with a 5' end A single-stranded DNA molecule with a FAM group, or a SHC ₆ group at the 5' end.

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, more preferably 15-21nt, and most preferably 16-18nt.

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 that binds to the reporter nucleic acid at positions 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 method is a 16% nucleic acid Urea-PAG electrophoresis detection method.

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 embodiment, the length of the fluorescent 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 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⁺ .

In another preferred embodiment, in the nucleic acid cleavage system, the concentration of divalent metal ions is 10 μM-3 mM, preferably 50 μM-2 mM, more preferably 100 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 ≤500 mM, preferably 20-500 mM.

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

In another preferred example, when in the reverse complementary region of the gDNA and the reporter nucleic acid, any base from the 4th to the 12th position, and the 14th to 15th position from the 5' end is present with the When reporting nucleic acid mismatches, the cleavage rate of the programmable endonuclease Argonaute is significantly reduced.

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 cleavage system, the concentration of the fluorescent 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 example, in the nucleic acid cutting system, the concentration of the programmable endonuclease Ago is 10nM-10μM, preferably 100nM-1μM, more preferably 300nM-500nM, and most preferably 400nM .

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 a second aspect of the present invention, a reaction system for enriching low-abundance target nucleic acid is provided, the reaction system is used to simultaneously perform polymerase chain reaction (PCR) and nucleic acid cleavage reaction on a nucleic acid sample, 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 PCR 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 20-200 nM, preferably 30-150 nM, more preferably 40-100 nM.

In another preferred embodiment, the reagents required for the PCR reaction include PCR Taq Master Mix (purchased from abm company (Applied Biological Materials (abm) Inc.)).

In another preferred embodiment, the reagents required for the PCR reaction further include a pair of amplification primers for the target nucleic acid.

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

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

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 embodiment, in the reaction system, the concentration of divalent metal ions is 50mM-2M, preferably 100mM-1M, more preferably 0.5mM.

In another preferred example, the reaction temperature (reaction program) of the reaction system is: 94°C, 5min; cycle number 10-30 (94°C, 30s; 52°C, 30s; 72°C, 20s); 72°C, 20s); 1min.

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

In another preferred example, 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 shown.

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

In another preferred example, 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 pair are shown in SEQ ID NO: 13 and 14, respectively shown.

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

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 a polymerase chain reaction (PCR) and a 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 PCR 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,

Among them, the ratio of F1b/F1a is ≥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 nucleic acid samples pre-amplified by PCR are PCR amplification products of 1-30, preferably 10-20, 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 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 PCR reaction" include: DNA polymerase.

In another preferred example, the "reagents required for PCR reaction" further include: dNTP, 1-5Mm Mg ²⁺ , PCR 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 (ie the first nucleic acid) corresponds to the targeting region of the non-target nucleic acid (ie the second nucleic acid).

In another preferred embodiment, in the amplification-cutting reaction system, the nucleic acid cutting tool enzyme is 30nM, and the DNA polymerase is a high temperature 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 ^™ Master Mix.

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 fragment, EGFR E746-A750 mutant sequence fragment, and EGFR L858R mutant sequence fragment.

In another preferred embodiment, 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 as shown in SEQ ID NOs: 9 and 10, respectively. Show;

And the probe sequence for detecting the wild-type EGFR sequence fragment is shown in SEQ ID NO:7, and the probe sequence for detecting the EGFR E746-A750 mutant sequence fragment is shown in 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 shown in SEQ ID NOs: 19 and 20, respectively;

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

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 the 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 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 Thermococcus eurythermalis; or a homologous analog thereof with the same or similar functions.

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_050002102.1; and

(ii) On the basis of the sequence shown in NCBI sequence number WP_050002102.1, 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 sequence number WP_050002102.1 ≥ 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-TeAgo protein, which has high-temperature nuclease activity, and is isolated from a deep-sea hydrothermal vent in the Guaymas Basin (depth: 2006.9m, ) Thermococcus eurythermalis is the starting strain.

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

In the present invention, the recombinant plasmid pET28a-TeAgo is constructed by excavating the gene of the TeAgo protein and comparing the sequences. The recombinant plasmid is transformed into Escherichia coli (DE3) to realize the heterologous expression of TeAgo, and is purified by Ni-NTA column. Ago protein produced by recombinant strains.

The molecular weight of the novel high-temperature Ago protein obtained by the invention is about 88kDa, and the enzyme can simultaneously use 5'-phosphorylated gDNA and 5'-hydroxylated gDNA to mediate the cleavage of single-stranded DNA and single-stranded RNA target nucleic acid, wherein 5'-phosphorylated gDNA-mediated cleavage of single-stranded DNA target nucleic acids is the most efficient. The optimum reaction temperature range is between 90-99.9 °C, and the thermal stability at 95 °C is good; Mn ²⁺ can be used as an active ion, and 50-2000μM Mn ²⁺ can keep it highly active; the enzyme is in NaCl The activity is higher when the concentration range is 20-500mM; the enzyme can use the 15nt-21nt 5'-P gDNA to generate a classical cleavage product at the 10-11 site; the enzyme has a strong preference for gDNA, only when using 5'-P modified gDNA at the 5' end has higher activity, other modifications such as 5'-OH, 5'-Biotin, 5'-NH ₂ , 5'-FAM, 5'-SH, etc. have lower activity ; The enzyme can distinguish single-point mismatch and double-point mismatch between target and gDNA, which will have certain application prospects in SNV gene detection. The enzyme can effectively enrich EGFR del E746-A750 and EGFR L858R mutant genes through the coupling reaction of "cleaving while PCR" using the "A-STAR" detection technology.

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 the phylogenetic analysis for TeAgo.

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

Figure 3 shows the results of the shear activity assay for TeAgo.

Figure 4 shows a graph of the results of the optimum temperature range required for the TeAgo reaction.

Figure 5 shows the resulting graphs of the thermal stability of TeAgo at 90°C (A) and 95°C (B).

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

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

Figure 8 shows a graph of the results for the NaCl temperature range that TeAgo can tolerate.

Figure 9 shows a graph of the results of TeAgo's preference for gDNA 5' end modification.

Figure 10 shows the results of differential cleavage performed by TeAgo for single-point mismatches at different sites between gDNA and Target.

Among them, A shows the design method and specific sequence of the gDNA in the single-point mismatch experiment; B shows the experimental results of the single-point mismatch.

Figure 11 shows the results of differential cleavage performed by TeAgo for the double-point mismatch between the 10-11 site between gDNA and Target.

Among them, A shows the design method and specific sequence of the gDNA in the double-point mismatch experiment; B shows the experimental results of the double-point mismatch.

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

Figure 13 shows the high-sensitivity detection and preferred enrichment results of TeAgo for EGFR-delE746-A750 low-abundance mutant DNA (5%, 1%, 0.1%) substrates.

Figure 14 shows a standard curve for the detection of EGFR L858R low-abundance mutant DNA substrates by the dual TaqMan probe method.

Figure 15 shows the high-sensitivity detection and preferred enrichment results of TeAgo for EGFR L858R low-abundance mutant DNA (5%, 1%, ) substrates.

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 TeAgo through in vitro expression, purification and isolation, and obtained its optimal reaction parameters through a large number of groping experiments, thereby providing a TeAgo-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., the sensitivity can reach 0.01%, the DNA amount of the sample can be as low as aM level, and the detection of low-abundance mutant genes in human liquid biopsy can be better performed. It 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 pathogen-infectious diseases (viruses, pathogenic bacteria) detection fields and other fields. The present invention has been completed on this basis. 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 Thermococcus eurythermalis", "nuclease Thermococcus eurythermalis", "TeAgo enzyme" are used interchangeably and refer to the enzymes described in the first aspect of the invention.

The wild-type TeAgo enzyme has the amino acid sequence shown in NCBI SEQ ID NO: WP_050002102.1.

The TeAgo enzymes of the present invention may also contain mutant forms that retain functional activity. Said mutant form may contain one or more amino acid residue substitutions, deletions, changes or insertions on the basis of the sequence shown in NCBI sequence number WP_050002102.1, 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_050002102.1 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 TeAgo enzyme.

"A-STAR" detection technology

The core of the present invention lies in the development of a new nucleic acid cutting tool enzyme TeAgo with high temperature stability with single-point nucleic acid recognition specificity, coupled with PCR reaction to realize the process of cutting-while-amplification, and establishing "A-STAR ( A go- mediated S pecific Target detection)” technology, the 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 this temperature, TeAgo will melt a pair of gDNA under the guidance of a specific design. Wild-type gene ssDNA cutting, that is, this process can specifically cut the wild-type gene while retaining the mutant gene; in the subsequent PCR annealing step, the designed primers are located at least 20 nt upstream and downstream of the SNV site of the target nucleic acid, so it is not Selectively combine wild-type genes and mutant genes; in the subsequent PCR extension step, since the wild-type gene has been cleaved at the mutation site, it cannot be used as a template for extension, while the mutant gene retains its original length, so it can be used as a template for Amplification. Since this TeAgo high-temperature-specific cleavage coupled with PCR amplification can be performed in each cycle of conventional PCR (20-35 cycles), cleavage-side-amplification is achieved to efficiently enrich low-abundance mutant genes .

The technical advantages are: 1) High temperature distinguishes shearing, easy to operate; 2) The gDNA sequence is paired with the target sequence, which has high specificity; 3) It can be designed for any target sequence without sequence preference; 4) A single enzyme can pair multiple nucleic acid targets Realize multiple detection; 5) Can be combined with multi-terminal detection technology.

The coupling reaction of "cleaving while PCR"

In the present invention, when the TeAgo-gDNA complex is used to carry out the coupling reaction of "cutting while PCR", the reaction can be carried out under suitable conditions using the corresponding cutting enzyme and the corresponding amplification enzyme, as long as the conditions are The cleavage enzymes and amplification enzymes described 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 TeAgo protein concentration in the enrichment reaction system: preferably 20-100nM;

③ 94 ℃ TeAgo-gDNA complex pre-processing time (Pre-processing time (minute)): preferably 3-10 minutes;

④The initial concentration of gDNAs in the enrichment reaction system: preferably 200-2000nM;

⑤ The molar concentration ratio between TeAgo protein and gDNAs: preferably 1:5 to 1:20;

⑥The cycle number of enrichment PCR cycle: preferably 10-30;

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 "cutting while PCR", and is used to simultaneously perform polymerase chain reaction on a nucleic acid sample. PCR and nucleic acid cleavage reactions to obtain amplification-cleavage reaction products.

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 PCR reaction and (ii) the nucleic acid cleavage system based on the programmable endonuclease Argonaute (Ago) of the present invention .

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 addition, the reagents required for carrying out the PCR 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 100-300 nM, preferably 150-250 nM, more preferably 200 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 50mM-2M, preferably 100mM-1M, more preferably 0.5mM.

In the process of enriching the target nucleic acid, preferably, the reaction temperature (reaction program) of the reaction system is: 94°C, 5min; cycle times 10-30 (94°C, 30s; 52°C, 30s; 72°C, 20s) ); 72°C, 1 min.

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, The polymerase chain reaction (PCR) and nucleic acid cleavage reaction are carried out in the amplification-cleavage reaction system to obtain the amplification-cleavage reaction product; wherein, the nucleic acid cleavage reaction is used for specific cleavage of non-target nucleic acid, but not cleavage The target nucleic acid; and, the amplification-cutting reaction system contains (i) reagents required for PCR reaction and (ii) the nucleic acid cutting system of the present invention; wherein, the target nucleic acid is amplified in the amplification - The abundance in the cleavage reaction product is F1b; wherein, the ratio of F1b/F1a is ≥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 E746-A750 mutant sequence fragment, or an EGFR L858R 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) Provides an Ago that can withstand ultra-high temperature and has good stability.

2) An Ago that can better distinguish mismatches between target and gDNA is provided.

3) A nucleic acid tool enzyme that can be applied to gene detection and successfully enriches low-abundance mutant DNA with high efficiency is provided.

4) A nucleic acid tool enzyme with gene manipulation potential is provided.

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 TeAgo 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. TeAgo was selected as a candidate enzyme, among which, TeAgo and The sequence similarity of the characterized PfAgo reached 33.02%. The amino acid sequence of TeAgo (WP_050002102.1) and the corresponding gene sequence encoding the protein (NZ_CP008887.1) 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 TeAgo protein

The above-mentioned TeAgo-pET28a prokaryotic expression plasmid was introduced into E.coli BL21(DE3) to obtain TeAgo-pET28a/E.coli BL21(DE3) prokaryotic expression strain. The expression strain E.coli BL21(DE3) containing the recombinant plasmid TeAgo-pET28a was inoculated into LB medium containing 50 μg/mL kanamycin, and cultured at 37°C and 220rpm on a shaker 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 TeAgo protein. The cells were collected by centrifugation, and the cells were resuspended in a resuspension buffer (containing 20 mM Tris-HCl, pH 8.0 or so, 500 mM NaCl), then the cells were disrupted by high pressure, and the supernatant was obtained by centrifugation. 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 concentration of purified target protein based on this, and store the protein in a -80°C refrigerator for later use. TeAgo protein was analyzed by SDS-PAGE electrophoresis.

The results are shown in Figure 2. The results showed that a clear band was obtained at 88.1kDa, which indicated that the target protein TeAgo had been purified.

Example 3: TeAgo Shearing 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: 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’

Prepare reaction buffer (containing 15mM Tris-HCl pH8.0, 250mM NaCl), add 0.5mM MnCl ₂ , 400nM TeAgo, 2μM synthetic gDNA or gRNA and 0.8μM 5' fluorescently modified MnCl 2 to the reaction buffer Sequence complementary single-stranded DNA or RNA target nucleic acid, react at 95°C for 15min, 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 showed that TeAgo could utilize 5'-OH and 5'-P modified gDNA to cleave ssDNA and ssRNA, respectively.

Example 4: Analysis of TeAgo Catalytic Properties

The enzymatic activity of TeAgo was investigated at different temperatures (60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C), and the final concentration was 0.5mM in the reaction buffer. MnCl2, _TeAgo with a final concentration of 400nM, 2μM synthetic gDNA and 0.8μM 60nt sequence complementary single-stranded DNA target nucleic acid were reacted at different temperatures for 15min, and the reaction products were detected by electrophoresis under 16% nucleic acid Urea-PAGE.

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

The thermal stability of TeAgo was determined at 90 °C and 95 °C, respectively, under the same conditions: first add MnCl ₂ , TeAgo and gDNA to the reaction buffer, and incubate at 90 °C and 95 °C for 0, 5 min, 10min, 15min, 20min, 25min, 30min. The reaction products were tested under the same conditions.

The results are shown in Figure 5. The results showed that TeAgo had good thermal stability at both 90 ℃ and 95 ℃, and the shearing activity did not decrease after incubation for 30 min.

The concentrations of TeAgo, 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, and the reaction was carried out at a reaction temperature of 90 ℃ for 15 minutes. , the effect of metal ions on enzyme activity was detected by electrophoresis under 16% nucleic acid Urea-PAGE.

The results are shown in 6A. The results show that _TeAgo utilizes only MnCl2 as the active metal ion.

The reaction system and conditions were unchanged, and different concentrations of MnCl ₂ were added: 25 μM, 50 μM, 100 μM, 250 μM, 500 μM, 1000 μM, 2000 μM, and the optimal MnCl ₂ concentration of TeAgo was determined under the mediation of the 5' phosphorylated guide strand.

The results are shown in 6B. The results show that _25-2000mM MnCl2 can keep TeAgo highly active.

11-30nt 5'phosphorylated gDNAs were designed to explore the effects of different lengths of gDNA on TeAgo enzymatic activity. MnCl ₂ with a final concentration of 0.5 mM, TeAgo with a final concentration of 400 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, and the reaction products were reacted at 95°C for 15 min, respectively. Electrophoretic detection was performed under 16% nucleic acid Urea-PAGE.

The results are shown in Figure 7. The results show that TeAgo can use 15-21nt gDNA for conventional cleavage between 10-11 sites; however, when the length of the gDNA increases to 30nt, TeAgo undergoes unconventional cleavage, resulting in the generation of multiple products.

Adjust the composition of the reaction buffer, and configure the reaction buffer with a final concentration of 15mM Tris-HCl pH8.0 and different concentrations of NaCl (20mM, 50mM, 100mM, 250mM, 500mM, 1000mM, 2000mM, 3000mM, 4000mM, 5000mM), other The reaction system was unchanged, and the reaction was performed at 95°C for 15 min, and electrophoresis was performed under 16% nucleic acid Urea-PAGE.

The results are shown in Figure 8. The results showed that when the NaCl concentration in the reaction buffer was 20-500 mM, TeAgo could maintain a high activity; when the NaCl was too high, the shearing activity of TeAgo was inhibited.

Design and synthesize phosphorylated gDNA with different 5' end modifications: 5'-P, 5'-OH, 5'-Biotin, 5'-NH ₂ , 5'-FAM, 5'-SH, the reaction system is unchanged, and MnCl is added _2. For gDNA and target DNA, the shearing efficiency of TeAgo under the mediation of 5' different modified guide strands was determined. The reaction was carried out at 95°C for 15 min, and electrophoresis was carried out under 16% nucleic acid Urea-PAGE.

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

Design wild-type genes in the range of 60-90nt, mutant genes 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, and MnCl ₂ is added , and mismatched gDNA and target DNA at different sites to determine the differential shearing effect of TeAgo. The reaction was carried out at 95°C for 15 min, and electrophoresis was carried out under 16% nucleic acid Urea-PAGE.

The principle and results of primer design are shown in Figure 10. The results show that TeAgo has a low tolerance for single-base mutations, and a single site mismatch can lead to a significant decrease in splicing activity.

Design wild-type genes in the range of 60-90nt, mutant genes with single-base mutation, and a series of gDNAs with continuous double-point mismatches with the target at positions 10-11. The reaction system is unchanged, adding MnCl ₂ , and Different sites of mismatched gDNA and target DNA were used to determine the differential shearing effect of TeAgo. The reaction was carried out at 95°C for 15 min, and electrophoresis was carried out under 16% nucleic acid Urea-PAGE.

The principle and results of primer design are shown in Figure 11. The results show that the 10-11 consecutive double-point mismatches have a greater impact on the activity of TeAgo; however, for the two single strands of EGFR L858R, there is still a gDNA of TeAgo that can distinguish WT and Mut genes, providing candidate gDNAs for subsequent Mut gene enrichment. .

Example 5: Enrichment of mutant dsDNA by TeAgo-gDNA complexes

Design specific amplification primers, gDNAs and detection probes for the sequence characteristics of the EGFR del E746-A750 gene fragment. The specific sequences are shown in Table 1 and Table 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 products were 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 1 nM 5.0%, 1.0%, 0.01% mut EGFR del E746-A750 samples.

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

The reaction system included 2×PCR Taq Master Mix, 40-100nM TeAgo, 200nM forward and reverse amplification primers each, template with a final concentration of 1nM, 800-2000nM forward and reverse gDNAs, and 0.5mM MnCl ₂ .

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

In this example, the TaqMan fluorescence quantitative PCR method was used to quantitatively analyze the enriched products. Firstly, the standard curve of double TaqMan probe method for detection of EGFR del E746-A750 low-abundance mutant DNA substrate was determined. The 157bp wild-type and 142bp mutant EGFR del E746-A750 genes were mixed at a ratio of 1:1, and were serially diluted to 1nM, 100pM, 10pM, 1pM, 100fM, 10fM, ddH ₂ O, and used as a standard curve, as shown in Figure 12 .

Taking the 20 μL system as an example, the conditions of the TaqMan-qPCR detection system are as follows: 2 × Vazyme Mix, wild-type probe 0.5 μM, mutant probe 0.5 μM, forward and reverse probe primers 0.25 μM, and diluted template 5 μL. The TaqMan-qPCR program was as follows: 95°C, 8 min; (95°C, 15s; 60°C, 40s).

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

The enrichment results were processed as shown in Figure 13. The results showed that for the EGFR del E746-A750 gene, TeAgo could efficiently enrich the Mut genes with an allele mutation frequency (VAF) of 5.0% and 1.0%; for the Mut genes with a VAF of 0.1%, TeAgo An enrichment of around 60% is also possible.

Example 7: Enrichment of EGFR L858R mutant dsDNA by TeAgo-gDNA complexes

First, perform gDNA screening: According to the sequence characteristics of the two strands of the EGFR L858R gene, design a wild-type gene in the range of 60-90nt, a mutant gene with a single base mutation, and a series of consecutive double-stranded genes with the target at positions 10-11. The point-mismatched gDNA, the reaction system was unchanged, MnCl ₂ , and the different-site mismatched gDNA and target DNA were added to determine the differential cleavage effect of TeAgo. The reaction was carried out at 95°C for 15 min, and electrophoresis was carried out under 16% nucleic acid Urea-PAGE. The results are shown in Figure 11 (ABCD above).

Design specific amplification primers, gDNAs and detection probes according to the sequence characteristics of the EGFR L858R gene fragment. See Table 3 and Table 4 for details.

table 3

Table 4

The 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 prepared samples were quantified using the Pikogreen dsDNA quantification kit (supersensitive) (compatible with Qubit 3.0) sold by Liji Bio. Templates were configured as 1 nM 5.0%, 1.0% mut EGFR L858R samples.

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

The reaction system included 2×PCR Taq Master Mix, 40-100nM TeAgo, 200nM forward and reverse amplification primers each, template with a final concentration of 1nM, 800-2000nM forward and reverse gDNA, and 0.5mM MnCl ₂ .

Example 8: Detection of wild-type and mutant DNA products after enrichment of EGFR L858R mutant gene

In this example, the TaqMan fluorescence quantitative PCR method was used to quantitatively analyze the enriched products. Firstly, the standard curve of double TaqMan probe method for detection of EGFR L858R low-abundance mutant DNA substrate was determined. The 148bp wild-type and 148bp mutant EGFR L858R genes were mixed at a ratio of 1:1 and serially diluted to 100pM, 10pM, 1pM, 100fM, 10fM, 1fM, 100aM, ddH ₂ O, and the standard curve is shown in Figure 14. Taking a 20 μL system as an example, the conditions of the TaqMan-qPCR detection system are as follows: 2×Vazyme Mix, wild-type probe 0.5 μM, mutant probe 0.5 μM, forward and reverse probe primers 0.25 μM, and diluted template 5 μL. The TaqMan-qPCR program was as follows: 95°C, 8 min; (95°C, 15s; 60°C, 40s).

The enrichment results were processed as shown in Figure 15. The results show that, for the EGFR L858R gene, TeAgo can efficiently enrich nearly 70% of the Mut gene with an allele mutation frequency (VAF) of 5.0%; for the Mut gene with a VAF of 1%, TeAgo can also perform a little enrichment. set.

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

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.
The nucleic acid cutting system of claim 1, wherein the programmable endonuclease Argonaute is derived from Thermococcus eurythermalis, and the programmable endonuclease Argonaute is a programmable endonuclease Dicer TeAgo.
The nucleic acid cutting system according to claim 1, wherein the working stability of the nucleic acid cutting system is 80-99.9°C.
The nucleic acid cutting system of claim 1, wherein the guide DNA is a single-stranded DNA molecule phosphorylated at the 5' end.
The nucleic acid cutting system according to claim 1, wherein the length of the guide DNA is 5-30nt, more preferably 15-21nt, and most preferably 16-18nt.
The nucleic acid cutting system according to claim 1, wherein the guide DNA and the reporter nucleic acid have a reverse complementary fragment.
The nucleic acid cutting system according to claim 1, wherein 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.
A reaction system for enriching low-abundance target nucleic acid, characterized in that the reaction system is used to simultaneously perform 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 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 PCR reaction and (ii) the nucleic acid cleavage system according to claim 1 .
The reaction system of claim 8, wherein the gDNA comprises 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.
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 a polymerase chain reaction (PCR) and a 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 PCR 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,

Among them, the ratio of F1b/F1a is ≥10.
The method of claim 10, wherein the target nucleic acid and the non-target nucleic acid differ by only one base.
The method of claim 10, wherein the gDNA in the nucleic acid cutting system forms a first complementary binding region with the nucleic acid sequence of the targeting region of the target nucleic acid (ie, the first nucleic acid); and the nucleic acid cutting system The gDNA in also forms a second complementary binding region with the nucleic acid sequence of the targeting region of the non-target nucleic acid (ie, the second nucleic acid).
The method of claim 12, wherein at least 2 unmatched base pairs are contained in the first complementary binding region, thereby causing the complex not to cleave the target nucleic acid; and in the second complementary binding region The region contains 1 mismatched base pair, causing the complex to cleave the non-target nucleic acid.
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 8 or a reagent for preparing the reaction system;

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

(ii) Instructions for use, said instructions describing the method of claim 10.
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.