Characterization and application of novel normal-temperature Argonaute protein

Abstract

The invention provides characterization and application of a novel normal-temperature Argonaute protein, and particularly provides a detection system for directly detecting a target nucleic acid molecule, the system comprises: (a) a guide gDNA or gRNA pair; (b) a normal-temperature Argonaute protein (Ago enzyme), wherein the normal-temperature Argonaute protein (Ago enzyme) is a normal-temperature Argonaute protein (Ago enzyme) which is used for guiding to shear target RNA (Ribonucleic Acid) by taking DNA (Deoxyribose Nucleic Acid) or RNA (Ribonucleic Acid) as a guide; (c) fluorescence report nucleic acid, the fluorescence report nucleic acid has a fluorophore and a quenching group, and the target nucleic acid molecule is target DNA or target RNA. After the detection system is optimized, the efficiency is greatly improved, the target belongs to RNA types which are less found at present, and a new enzyme resource is expected to be provided for in-vivo RNA editing and nucleic acid detection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the characterization and application of a novel room-temperature Argonaute protein. Background Art

[0002] Argonaute (Ago) proteins were 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 the host against invading RNA viruses and protecting genome integrity. Currently reported Ago proteins are mainly divided into two categories: eukaryotic Ago (eAgo) and prokaryotic Ago (pAgo).

[0003] 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. Unlike the CRISPR / Cas system, prokaryotic Ago does not require a PAM sequence when cleaving the target nucleic acid chain. It can bind to the guide DNA or RNA that is complementary to the target nucleic acid and cleave at any complementary corresponding position of the target.

[0004] Most of the pAgos that have been characterized so far that can cut double-stranded DNA are derived from thermophilic bacteria, and their double-stranded DNA cutting activity is very low at moderate or room temperature (20-50°C), which limits the wide application of Ago in gene editing and gene detection. Although many researchers have worked together to explore the mechanism and biological role of pAgos, other unknown room-temperature pAgos in the Ago protein library with huge biological potential still need further exploration.

[0005] Therefore, there is an urgent need in this field to break through the limitations of traditional nucleic acid RNA detection methods in terms of time, cost and operational difficulty, and to establish a nucleic acid detection method with high sensitivity, good specificity, rapidity and efficiency. Summary of the invention

[0006] The purpose of the present invention is to break through the limitations of traditional nucleic acid RNA detection methods in terms of time, cost and operational difficulty, and to establish a nucleic acid detection method with high sensitivity, good specificity, rapidity and high efficiency.

[0007] In a first aspect of the present invention, a detection system for directly detecting a target nucleic acid molecule is provided, the system comprising:

[0008] (a) a guide gDNA pair or a guide gRNA pair;

[0009] (b) a room-temperature Argonaute protein (Ago enzyme), wherein the room-temperature Argonaute protein (Ago enzyme) is a room-temperature Argonaute protein (Ago enzyme) that uses DNA or RNA as a guide to guide the cleavage of a target RNA;

[0010] (c) a fluorescent reporter nucleic acid having a fluorescent group and a quencher group;

[0011] Wherein, the target nucleic acid molecule is a target DNA or a target RNA.

[0012] In another preferred embodiment, in the detection system, the target DNA is transcribed to form a target RNA, which is recognized and cleaved by the room temperature Argonaute protein (Ago enzyme).

[0013] In another preferred embodiment, in the detection system, the target RNA is reverse transcribed-transcribed to form a target RNA, which is recognized and cleaved by the room temperature Argonaute protein (Ago enzyme).

[0014] In another preferred embodiment, the room temperature Argonaute protein (Ago enzyme) has the ability to guide the cleavage of target RNA using DNA or RNA as a guide.

[0015] In another preferred embodiment, the room-temperature Argonaute protein (Ago enzyme) is derived from the room-temperature halophilic bacteria Haloferax_profundi, Haloferax larsenii, Haloferax alexandrinus, Haloferax alexandrinus, Halocatena pleomorpha, Halorussus marinus, Halorubrum sp.48-1-W, Haloarchaeobiussalinus, Halosimplex salinum, Natronomonas gomsonensis, Halogeometricumborinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculus aciditolerans, Halalkaliarchaeum desulfuricum, and Halosimplexpelagicum.

[0016] In another preferred embodiment, the room-temperature Argonaute protein (Ago enzyme) is selected from the group consisting of Haloferax_profundi (HpAgo), Haloferax larsenii, Haloferax alexandrinus (HaleAgo), Haloferaxalexandrinus (HaalAgo), Halocatena pleomorpha (HaplAgo), Halorussus marinus (HamaAgo), Halorubrum sp.48-1-W (HaspAgo enzyme), Haloarchaeobius salinus (HalosaAgo), Halosimplex salinum, Natronomonas gomsonensis, Halogeometricum borinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculusaciditolerans, Halalkaliarchaeum desulfuricum, Halosimplex pelagicum and mutants thereof.

[0017] In another preferred embodiment, the room-temperature Argonaute protein (Ago enzyme) includes Haloferax_profundi (HpAgo) and a room-temperature Argonaute protein (Ago enzyme) having a homology of more than 85% (preferably more than 90%, more preferably more than 95%, more preferably more than 98%) compared with Haloferax_profundi (HpAgo).

[0018] In another preferred embodiment, the amino acid sequence of the room temperature Argonaute protein (Ago enzyme) is shown in SEQ ID NO.1.

[0019] In another preferred embodiment, the room temperature Argonaute protein (Ago enzyme) has an amino acid sequence selected from the following group:

[0020] (i) having the amino acid sequence shown in SEQ ID NO.: 1;

[0021] (ii) a polypeptide derived from (i) having the activity of the normal temperature Argonaute protein (Ago enzyme) formed by replacing, deleting or adding one or several (e.g., 1-10) amino acid residues of the amino acid sequence as shown in SEQ ID NO.: 1; or

[0022] (iii) a polypeptide derived from (i) having an amino acid sequence with a homology of ≥85% (preferably ≥90%, more preferably ≥95% or ≥98%) to the amino acid sequence shown in SEQ ID NO.: 1 and having the activity of the said room temperature Argonaute protein (Ago enzyme).

[0023] In another preferred example, the nucleotide sequence of the guide gDNA is as shown in SEQ ID NO:4.

[0024] In another preferred example, the nucleotide sequence of the guide gRNA is as shown in SEQ ID NO:5.

[0025] In another preferred example, the guide gDNA pair includes a primary guide gDNA and a secondary guide gDNA.

[0026] In another preferred embodiment, the guide gRNA pair includes a primary guide gRNA and a secondary guide gRNA.

[0027] In another preferred embodiment, the guide gDNA or gRNA is a single-stranded DNA or single-stranded RNA molecule.

[0028] In another preferred embodiment, the guide gDNA comprises a single-stranded DNA molecule with a 5' end phosphorylated or hydroxylated.

[0029] In another preferred embodiment, the guide gRNA comprises a single-stranded RNA molecule with a phosphorylated or hydroxylated 5' end.

[0030] In another preferred embodiment, the guide gDNA or gRNA is a 5'-phosphorylated single-stranded DNA or single-stranded RNA molecule.

[0031] In another preferred embodiment, the guide gDNA and guide gRNA have complementary fragments to the fluorescent reporter nucleic acid.

[0032] In another preferred embodiment, the length of the guide gDNA is 8-60 nt, preferably 10-30 nt.

[0033] In another preferred embodiment, the length of the guide gRNA is 8-60 nt, preferably 10-30 nt.

[0034] In another preferred embodiment, the first nucleotide at the 5' end of the guide gDNA is phosphorylated or hydroxylated thymine (T).

[0035] In another preferred embodiment, the first nucleotide at the 5' end of the guide gRNA is phosphorylated or hydroxylated uracil (U).

[0036] In another preferred embodiment, the number of the primary guide gDNAs or gRNAs is one or more pairs.

[0037] In another preferred embodiment, the number of the secondary guide gDNAs or gRNAs is one or more.

[0038] In another preferred embodiment, the primary guide gDNAs or gRNAs are complementary to the target RNA.

[0039] In another preferred embodiment, the Ago enzyme includes wild-type and mutant Ago.

[0040] In another preferred embodiment, the mutation sites corresponding to the different typings of the target RNA are at positions 2-16 of the primary guide gDNA or gRNA, preferably, at positions 2-5 of the primary guide gDNA or gRNA.

[0041] In another preferred embodiment, the detection system further contains (d) reagents required for reverse transcription and transcription.

[0042] In another preferred embodiment, the reagents required for reverse transcription include reverse transcriptase: II reverse transcriptase, RNase Inhibitor, template, dNTP Mix, reverse transcription primer, 5×ProtoScript II Buffer, DTT, reverse transcription reporter, MnCl2, reverse transcription Guide A&B, HpAgo, Nuclease-free Water 0.0.

[0043] In another preferred embodiment, the reagents required for transcription include: Nuclease-free Water, dNTP Buffer Mix, purified product, and T7 RNA Polymerase.

[0044] In another preferred embodiment, the reagents required for reverse transcription further include: dNTP, DTT and reverse transcription reaction buffer.

[0045] In another preferred embodiment, the reverse transcriptase is AMV having RNA-guided DNA polymerase activity.

[0046] In another preferred embodiment, the amount of RNA used as a template during reverse transcription is 10 -2 -10 6 nM (preferably, 10 -1 —10 5 nM, more preferably, 1-10 4 nM).

[0047] In another preferred embodiment, when the fluorescent reporter nucleic acid is cleaved, the cleavage can be detected by urea polyacrylamide electrophoresis.

[0048] In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5' end or the 3' end of the fluorescent reporter nucleic acid.

[0049] In another preferred embodiment, the fluorescent group and the quencher group are respectively located on both sides of the complementary region between the fluorescent reporter nucleic acid and the guide gDNA or guide gRNA.

[0050] In another preferred embodiment, the fluorescent groups include FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, and Texas Red-X.

[0051] In another preferred embodiment, the quenching group includes: BHQ, TAMRA, DABCYL, and DDQ.

[0052] In another preferred embodiment, the length of the fluorescent reporter nucleic acid is 10-100 nt, preferably 20-70 nt, more preferably 30-60 nt, and more preferably 40-50 nt.

[0053] In another preferred embodiment, the working temperature of the room temperature Argonaute protein (Ago enzyme) is 5-60°C, preferably 10-45°C, and more preferably 25-37°C.

[0054] In another preferred embodiment, the action time of the room temperature Argonaute protein (Ago enzyme) is ≥5 minutes, preferably ≥15 minutes, and more preferably ≥30 minutes.

[0055] In another preferred embodiment, the detection system further comprises: (e) divalent metal ions.

[0056] In another preferred embodiment, the divalent metal ion is selected from the following group: Mg 2+ , Mn 2+ , Fe 2+ 、Co 2+ , Cu 2+ 、Ni 2+ 、Zn 2+ , Ca 2+ , or a combination thereof.

[0057] In another preferred embodiment, the divalent metal ion is selected from the following group: Co 2+ , Mn 2+ , or a combination thereof.

[0058] In another preferred embodiment, the magnesium ions come from magnesium chloride or magnesium sulfate, preferably magnesium chloride.

[0059] In another preferred embodiment, the manganese ions come from manganese chloride.

[0060] In another preferred embodiment, the Fe 2+ Derived from ferrous chloride.

[0061] In another preferred embodiment, the Co 2+ From cobalt chloride.

[0062] In another preferred embodiment, the Cu 2+ From copper sulfate.

[0063] In another preferred embodiment, the Ni 2+ From nickel chloride.

[0064] In another preferred embodiment, the Zn 2+ From zinc chloride.

[0065] In another preferred embodiment, the Ca 2+ From calcium chloride.

[0066] In another preferred embodiment, in the detection system, the concentration of the divalent metal ions is 0mM-8M, preferably 500mM-8M, and more preferably 1M-8M.

[0067] In another preferred embodiment, the Mn 2+ The concentration is ≥50 μM, preferably, ≥250 μM, more preferably, 1000 μM-80000 μM.

[0068] In another preferred embodiment, the Co 2+ The concentration is 0.25M-8M, preferably, 1.5M-2M.

[0069] In another preferred embodiment, the detection system further comprises: (f) a buffer solution.

[0070] In another preferred embodiment, in the buffer solution, the concentration of NaCl is ≤2000 mM, preferably ≤250 mM, more preferably ≤100 mM, and more preferably ≤50 μM.

[0071] In another preferred embodiment, the pH value of the buffer solution is 4-10, preferably 6-8.

[0072] In another preferred embodiment, the detection system further contains a target nucleic acid molecule to be detected.

[0073] In another preferred embodiment, the target nucleic acid molecule or its reverse transcription-transcription product is cleaved by the Ago enzyme to produce a secondary guide gRNA.

[0074] In another preferred embodiment, the length of the secondary guide gRNA is 8-60 nt, preferably, 10-30 nt.

[0075] In another preferred embodiment, the sequence of the secondary guide gRNA is complementary to that of the fluorescent reporter nucleic acid.

[0076] In another preferred embodiment, after the secondary guide gRNA binds complementary to the sequence of the fluorescent reporter nucleic acid, it guides the Ago enzyme to cut the fluorescent reporter nucleic acid, thereby generating a detectable signal (such as fluorescence).

[0077] In another preferred embodiment, the concentration of the target nucleic acid molecule to be detected in the detection system is 0.01-20uM, preferably 0.05-1uM, and more preferably 0.08-0.5uM.

[0078] In another preferred embodiment, in the detection system, the concentration of the room temperature Argonaute protein (Ago enzyme) is 0.1-20uM, preferably, 2-4μM.

[0079] In another preferred embodiment, in the detection system, the concentration of the guide DNA or RNA is 0.025-20uM, preferably 0.1-1uM, and more preferably 0.2-0.8uM.

[0080] In another preferred embodiment, in the detection system, the molar ratio of the fluorescent reporter nucleic acid to the target nucleic acid molecule is 1:1.8 to 1:100000, preferably 1:36000.

[0081] In another preferred embodiment, in the detection system, the molar ratio of the target nucleic acid molecule to be detected, the guide DNA or RNA, and the room temperature Argonaute protein (Ago enzyme) is 1:(0.05-30):(0.5-200), preferably 1:(1-10):(5-50), and more preferably 1:4-6:20-40.

[0082] In another preferred embodiment, in the detection system, the concentration of the fluorescent reporter nucleic acid is 50-3000 nM.

[0083] In another preferred embodiment, the target RNA includes single-stranded RNA and double-stranded RNA.

[0084] In another preferred embodiment, the target DNA includes single-stranded DNA and double-stranded DNA.

[0085] In another preferred embodiment, the target DNA or RNA each independently includes a target DNA or RNA derived from a plant, an animal, a microorganism, a virus, or a combination thereof.

[0086] In another preferred embodiment, the target DNA or RNA is independently artificially synthesized or naturally occurring DNA or RNA.

[0087] In another preferred embodiment, the target DNA or RNA each independently includes wild-type or mutant DNA or RNA.

[0088] In another preferred embodiment, the detection system is a one-step detection system.

[0089] A second aspect of the present invention provides a kit for directly detecting a target nucleic acid molecule, the kit comprising:

[0090] (i) the detection system according to the first aspect of the present invention or a reagent for preparing the detection system; and

[0091] (ii) instructions for use, wherein the instructions describe a method for directly detecting a target nucleic acid molecule using the detection system.

[0092] In another preferred embodiment, the kit further comprises reagents required for transcription or reverse transcription-transcription.

[0093] In another preferred embodiment, the kit further comprises a divalent metal ion solution.

[0094] In another preferred embodiment, the kit comprises:

[0095] (a) a first container and a guide gDNA or guide gRNA located in the first container;

[0096] (b) a second container and a room-temperature Argonaute protein (Ago enzyme) located in the second container, wherein the room-temperature Argonaute protein (Ago enzyme) is a room-temperature Argonaute protein (Ago enzyme) that uses DNA or RNA as a guide to guide the cleavage of target RNA; and

[0097] (c) a third container and a fluorescent reporter nucleic acid located in the third container.

[0098] In another preferred embodiment, the kit further comprises:

[0099] (d) a fourth container and reagents required for performing transcription or reverse transcription-transcription located in the fourth container.

[0100] In another preferred embodiment, the kit further comprises:

[0101] (f) a fifth container and a divalent metal ion solution located in the fifth container.

[0102] The third aspect of the present invention provides a method for directly detecting whether a target nucleic acid molecule is present in a sample, comprising the following steps:

[0103] (a) providing the detection system for detecting target nucleic acid molecules according to the first aspect of the present invention; and

[0104] (b) reacting the detection system with a sample to be detected at a certain temperature to form a first reaction solution;

[0105] (c) performing fluorescence detection on the first reaction solution to obtain a fluorescence signal value;

[0106] Among them, if a fluorescence signal value is detected in the first reaction solution, it means that a target nucleic acid molecule exists in the sample; and if no fluorescence signal value is detected in the first reaction solution, it means that no target nucleic acid molecule exists in the sample, and the target nucleic acid molecule is a target DNA or a target RNA.

[0107] In another preferred embodiment, the sample to be detected is a sample obtained without amplification.

[0108] In another preferred embodiment, the sample to be tested includes nucleic acid from a test sample, wherein the test sample is selected from the following group: blood, cells, serum, saliva, body fluid, plasma, urine, prostatic fluid, bronchial lavage fluid, cerebrospinal fluid, gastric juice, bile, lymph fluid, peritoneal fluid and feces, or a combination thereof.

[0109] In another preferred embodiment, the sample to be detected includes a nucleic acid sample directly cleaved by heating, a nucleic acid sample directly treated with a protease, an extracted nucleic acid sample, a nucleic acid sample pre-amplified by PCR, or any sample containing nucleic acid.

[0110] In another preferred embodiment, the method is used to detect whether the nucleic acid at the target site has a SNP, a point mutation, a deletion, and / or an insertion.

[0111] In another preferred embodiment, the step (b) includes the step of transcribing or reverse transcription-transcription of the sample to be detected.

[0112] In another preferred embodiment, the fluorescence detection in step (c) is performed using a qPCR instrument, an enzyme-labeled instrument or a fluorescence spectrophotometer.

[0113] In another preferred embodiment, the method is an in vitro method.

[0114] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0115] In another preferred embodiment, the method is a one-step method.

[0116] 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 below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 The evolutionary tree of HpAgo and reported prokaryotic Ago is shown. As can be seen from the figure, HpAgo is most closely related to NgAgo among the reported prokaryotic Ago, and the sequence similarity is only about 26%.

[0118] Figure 2 The SDS-PAGE electrophoresis results of HpAgo expressed by the recombinant strain are shown, wherein M is a protein marker; 1 is the HpAgo purified solution.

[0119] Figure 3 The sequence alignment of HpAgo and reported prokaryotic Agos is shown. As can be seen from the figure, HpAgo, like other prokaryotic Agos, also contains a DEDX active center that exerts cleavage activity.

[0120] Figure 4 The figure shows the designed and synthesized FAM-labeled 45nt ssDNA / ssRNA target sequence (target) and the complementary 21nt 5'P / OH-modified DNA / RNA guide sequence (guide).

[0121] Figure 5 The figure shows the cleavage preference of purified HpAgo for the guide sequence (guide) and the target sequence (Target). As can be seen from the figure, HbAgo can use 5'P gDNA / gRNA to cleave the ssDNA complementary to it at 25°C.

[0122] Figure 6 The optimal temperature for HpAgo to exert cleavage activity under the guidance of two different gDNA / gRNAs is shown. Under the guidance of 5'PgDNA, the optimal reaction temperature is between 25℃-37℃; under the guidance of 5'P gRNA, the optimal reaction temperature is between 20℃-37℃.

[0123] Figure 7 The figure shows the preference of HpAgo for divalent metal ions under the guidance of two different gDNA / gRNA. As can be seen from the figure, HpAgo requires divalent metal ions such as Mn2+ and Co2+ to exert its cleavage activity.

[0124] Figure 8The figure shows the effect of Mn2+ concentration on HpAgo cleavage activity under the guidance of two different gDNA / gRNA. As can be seen from the figure, when the Mn2+ ion concentration is in the range of 500mM to 5M, the HpAgo enzyme activity remains at a high level.

[0125] Fig. 9 The effect of Co2+ concentration on HpAgo cleavage activity under the guidance of two different gDNA / gRNA is shown. When the Co2+ concentration ranges from 1M to 1.5M, the HpAgo enzyme activity remains at a high level.

[0126] Fig.10 The figure shows the tolerance of HpAgo to NaCl under the guidance of two different gDNA / gRNA. As can be seen from the figure, the enzyme is intolerant to NaCl, which inhibits the activity of PbAgo in cleaving ssDNA.

[0127] Fig.11 The effect of two different gDNA / gRNA guide lengths on HpAgo activity was shown. The results showed that HpAgo could cleave ssRNA under the guidance of a guide of 10-30nt in length.

[0128] Fig.12 The results show that HpAgo has no preference for the 5-terminal base of the guide.

[0129] Fig.13 The effect of introducing single mismatches between guide and target 2-16 on HpAgo activity is shown. As can be seen from the figure, when HpAgo is guided by 5'P gDNA, mismatches in the seed region (2-5) of the guide will inhibit the activity of HpAgo, and when HpAgo is guided by 5'P gRNA, mismatches in the seed region (2-5) and 16 of the guide will inhibit the activity of HpAgo.

[0130] Fig.14 The figure shows the effect of introducing double mismatches between guide and target 2-16 on HpAgo activity. As can be seen from the figure, HpAgo activity is significantly reduced overall. Under the guidance of 5'P gDNA, mismatches in the seed region (2-8) and 12-16 of the guide will inhibit the activity of HpAgo. DETAILED DESCRIPTION

[0131] After extensive and in-depth research, the inventors have developed for the first time a nuclease that targets target DNA or target RNA and has broad application prospects. Specifically, the present invention obtains the nuclease HpAgo by in vitro expression and purification and separation, and explores through a large number of experiments to obtain its optimal reaction parameters. The experimental results show that the optimal reaction temperature of the nuclease HpAgo of the present invention is between 20°C-37°C; the concentration of divalent metal ions in the range of 500mM to 3M can significantly promote the shearing activity of HpAgo, and the present invention also unexpectedly found that the nuclease HpAgo of the present invention is intolerant to NaCl. In addition, the reaction system of the present invention has greatly improved efficiency after optimization, and the target belongs to a type of RNA that is currently less discovered, which is expected to provide new enzyme resources for in vivo RNA editing and nucleic acid detection. On this basis, the inventors completed the present invention.

[0132] the term

[0133] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below.

[0134] There are clear definitions, and all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions may vary. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0135] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0136] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not occur.

[0137] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0138] As used herein, "transduction", "transfection", "transformation" or other terms used herein refer to the process of transferring exogenous polynucleotides to host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to introduce exogenous polynucleotides into host cells (e.g., E. coli).

[0139] As used herein, "gene expression" or "expression" refers to the process of gene transcription, translation and post-translational modification to produce the RNA or protein product of a gene.

[0140] As used herein, "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and analogs. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide used herein refers to single-stranded and double-stranded molecules that are interchangeable. Unless otherwise indicated, the polynucleotides in any embodiment described herein include double-stranded forms and two complementary single strands that are known or predictable to constitute the double-stranded form.

[0141] Conservative amino acid substitutions are known in the art. In some embodiments, the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and / or phenylalanine, tryptophan and tyrosine; methionine and cysteine. In addition, the present invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.

[0142] 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 specific application for which the invention is used. 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 of the embodiments of the invention is not limited to the scope specifically described and claimed.

[0143] Definitions and all definitions used herein should be understood to control over dictionary definitions or definitions in documents incorporated by reference.

[0144] All references, patents, and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, in some cases including in their entirety.

[0145] It should be understood that for any method described herein comprising more than one step, the order of the steps is not necessarily limited to the order described in the examples.

[0146] As used herein, the terms "the detection system of the present invention" and "the nucleic acid detection system based on normal temperature Argonaute protein (Ago enzyme)" are used interchangeably to refer to the detection system described in the first aspect of the present invention.

[0147] As used herein, the terms "the detection method of the present invention" and "the nucleic acid detection method based on normal temperature Argonaute protein (Ago enzyme)" are used interchangeably to refer to the detection method described in the third aspect of the present invention.

[0148] As used herein, the terms "normal temperature Argonaute protein (Ago enzyme)", "gene editing enzyme", and "Ago enzyme" are used interchangeably to refer to the enzyme described in the first aspect of the present invention.

[0149] As used herein, the term "secondary cleavage" refers to the fact that in the detection method of the present invention, in the presence of primary guide gDNAs or guide gRNAs, the room temperature Argonaute protein (Ago enzyme) of the present invention cleaves the target DNA sequence (such as single-stranded, double-stranded DNA) formed by reverse transcription of the target RNA or the target RNA sequence (such as single-stranded, double-stranded RNA) formed by reverse transcription-transcription of the target RNA, or directly cleaves the target RNA (such as single-stranded, double-stranded RNA) to form a new 5'phosphorylated nucleic acid sequence (secondary guide gDNAs or secondary guide gRNAs); then, the secondary guide gDNA or secondary guide gRNA continues to guide the Ago enzyme to cleave the fluorescent reporter nucleic acid complementary to the secondary guide gDNAs or secondary guide gRNAs under the action of the Ago enzyme. This specific cleavage (first cleavage) of the target RNA or DNA formed by the target nucleic acid sequence or directly targeting the target nucleic acid sequence, and then the specific cleavage of the fluorescent reporter nucleic acid (second cleavage) is defined as "secondary cleavage". In the present invention, both the first cleavage and the second cleavage are specific cleavages.

[0150] Ago enzyme

[0151] Argonaute proteins are widely present in all areas of life and can bind to siDNA or siRNA guide chains to specifically silence or cleave complementary nucleic acid target chains. They can act as nucleases in vitro and play a host defense role in vivo.

[0152] In the present invention, the preferred Ago enzyme is a room temperature Argonaute protein (Ago enzyme) that uses DNA or RNA as a guide to cleave the target RNA, and more preferably, is selected from the following group: (Haloferax_profundi)

[0153] (HpAgo), Haloferax larsenii, Haloferax alexandrinus (HaleAgo), Haloferaxalexandrinus (HaalAgo), Halocatena pleomorpha (HaplAgo), Halorussus marinus (HamaAgo), Halorubrum sp.48-1-W (HaspAgo enzyme), Haloarchaeobius salinus (HalosaAgo), Halosimplex salinum, Natronomonas gomsonensis, Halogeometricum borinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculusaciditolerans, Halalkaliarchaeum desulfuricum, Halosimplex pelagicum and their mutants.

[0154] In another preferred embodiment, the room temperature Argonaute protein (Ago enzyme) has an amino acid sequence selected from the following group:

[0155] (i) having the amino acid sequence shown in SEQ ID NO.: 1;

[0156] (ii) a polypeptide derived from (i) having the activity of the normal temperature Argonaute protein (Ago enzyme) formed by replacing, deleting or adding one or several (e.g., 1-10) amino acid residues of the amino acid sequence as shown in SEQ ID NO.: 1; or

[0157] (iii) a polypeptide derived from (i) having an amino acid sequence with a homology of ≥85% (preferably ≥90%, more preferably ≥95% or ≥98%) to the amino acid sequence shown in SEQ ID NO.: 1 and having the activity of the said room temperature Argonaute protein (Ago enzyme).

[0158] As used herein, the terms "nuclease Haloferax_profundi" and "HpAgo enzyme" are used interchangeably, the gene length is 2694 bp, the amino acid sequence consists of 895 amino acids, and the molecular weight is approximately 98.72 kDa.

[0159] The enzymatic cleavage characteristics of the room temperature Argonaute protein (Ago enzyme) (such as HpAgo enzyme) are: the enzyme can use the 5' phosphorylated oligonucleotide as a guide ssDNA / ssRNA to guide the enzyme to accurately cleave the target RNA sequence at room temperature; the cleavage site is located at the phosphodiester bond between the target RNA (ssRNA) corresponding to the 10th and 11th nucleotides of the guide ssDNA. Generally, the preferred working temperature of the HpAgo enzyme is 20-37°C.

[0160] The wild-type HpAgo enzyme has the amino acid sequence shown in SEQ ID NO:1.

[0161] (SEQ ID NO:1)

[0162] The HpAgo enzyme of the present invention may also include a mutant form thereof that has lost its functional activity. The mutant form may contain one or more amino acid residues substituted on the basis of the sequence shown in SEQ ID NO: 1; and the obtained amino acid sequence has a sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) with the sequence shown in SEQ ID NO: 1; and the obtained amino acid sequence lacks the same or similar function as the wild-type HpAgo enzyme.

[0163] Guide gDNA pair or guide gRNA pair

[0164] In the detection system and detection method of the present invention, a core component is a guide gDNA pair or a guide gRNA pair.

[0165] In the present invention, the preferred guide gDNAs are oligonucleotides with a length of 8-60 nt, and the first nucleotide of the 5' region is a phosphorylated or hydroxylated thymine (T).

[0166] In the present invention, the preferred guide gRNAs are all oligonucleotides with a length of 8-60 nt, and the first nucleotide of the 5' is a phosphorylated or hydroxylated modified uracil (U).

[0167] Testing system

[0168] As used herein, the terms "detection system of the present invention" and "nucleic acid detection system based on Argonaute protein" are used interchangeably and refer to the detection system for detecting target nucleic acid molecules described in the first aspect of the present invention. The detection system of the present invention is based on the reaction conditions provided by the present invention that are suitable for the functional activity of the Ago enzyme of the present invention (especially HpAgo enzyme) for detection.

[0169] In a preferred embodiment, the present invention provides a detection system for detecting a target nucleic acid molecule, comprising:

[0170] (a) Guide ssDNA / ssRNA pairs;

[0171] (b) a room-temperature Argonaute protein (Ago enzyme), wherein the room-temperature Argonaute protein (Ago enzyme) is a room-temperature Argonaute protein (Ago enzyme) that uses DNA / RNA as a guide to guide the cleavage of target RNA;

[0172] (c) a fluorescent reporter nucleic acid, wherein the fluorescent reporter nucleic acid carries a fluorescent group and a quencher group, and the length of the reporter nucleic acid is preferably 45 nt, 78 nt and 352 nt;

[0173] Wherein, the target nucleic acid molecule is target DNA or RNA.

[0174] In the detection system of the present invention, the working temperature of the nuclease is 10-45°C, preferably 20-37°C, and more preferably 25-37°C.

[0175] Preferably, the guide DNA / RNA is a single-stranded DNA / RNA molecule with 5' end phosphorylation. Preferably, the guide DNA / RNA has a length of 8-35 nt.

[0176] Preferably, the reporter nucleic acid is a single-stranded RNA (ssRNA). In one embodiment, when the reporter nucleic acid is cleaved, the cleavage can be detected by electrophoresis.

[0177] In another embodiment, the fluorescent group and quencher group of the fluorescent reporter nucleic acid are independently located at the 5' end and 3' end of the fluorescent reporter nucleic acid; preferably, the fluorescent group and quencher group are located on both sides of the complementary region between the fluorescent reporter nucleic acid and the guide DNA / RNA. In another preferred embodiment, the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof.

[0178] In another preferred embodiment, the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.

[0179] The length of the reporter nucleic acid is 10-100nt, preferably 20-70nt, more preferably 30-60nt, more preferably 40-50nt, such as 45nt.

[0180] The detection system of the present invention may also include divalent metal ions. The divalent metal ions are selected from the following group: Mg2+, Mn2+, Fe2+, Co2+, Cu2+, Ni2+, Zn2+, Ca2+, or a combination thereof. Preferably, they are: Co2+, Mn2+. In the detection system, the concentration of the divalent metal ions is 0mM-5M, preferably 500mM-5M, and more preferably 1M-5M.

[0181] In one embodiment, in the detection system of the present invention, the concentration of NaCl is ≤

[0182] 2000mM, preferably ≤250mM, more preferably ≤100mM.

[0183] Detection Methods

[0184] As used herein, the terms "the detection method of the present invention" and "the nucleic acid detection method based on Argonaute protein" are used interchangeably to refer to the detection method described in the third aspect of the present invention.

[0185] In the present invention, a method for directly detecting whether a target molecule exists in a sample is provided, comprising the following steps:

[0186] (a) providing the detection system for detecting target nucleic acid molecules according to the first aspect of the present invention; and

[0187] (b) reacting the detection system with a sample to be detected at a certain temperature to form a first reaction solution;

[0188] (c) performing fluorescence detection on the first reaction solution to obtain a fluorescence signal value;

[0189] Among them, if a fluorescence signal value is detected in the first reaction solution, it means that a target nucleic acid molecule exists in the sample; and if no fluorescence signal value is detected in the first reaction solution, it means that no target nucleic acid molecule exists in the sample, and the target nucleic acid molecule is a target DNA or a target RNA.

[0190] In a preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0191] In one embodiment, the detection in step (c) may include: using electrophoresis to identify the length of the reporter nucleic acid, thereby determining whether the reporter nucleic acid is cleaved. If a shorter nucleic acid product is generated, it indicates that the reporter nucleic acid is cleaved by Ago in the detection system.

[0192] In another embodiment, if the reporter nucleic acid in the detection system carries a fluorescent group and / or a quenching group, then the detection in step (c) includes: detecting using an enzyme reader or a fluorescence spectrophotometer.

[0193] In one embodiment of the invention, the method is an in vitro method.

[0194] In another embodiment, the method is non-diagnostic and non-therapeutic.

[0195] Reagent test kit

[0196] The present invention provides a kit for directly detecting a target nucleic acid molecule, the kit comprising:

[0197] (i) the detection system according to the first aspect of the present invention or a reagent for preparing the detection system; and

[0198] (ii) instructions for use, wherein the instructions describe a method for directly detecting a target nucleic acid molecule using the detection system.

[0199] Typically, the kit includes:

[0200] (a) a first container and a guide gDNA or guide gRNA located in the first container;

[0201] (b) a second container and a room-temperature Argonaute protein (Ago enzyme) located in the second container, wherein the room-temperature Argonaute protein (Ago enzyme) is a room-temperature Argonaute protein (Ago enzyme) that uses DNA or RNA as a guide to guide the cleavage of target RNA; and

[0202] (c) a third container and a fluorescent reporter nucleic acid located in the third container.

[0203] In another preferred embodiment, the kit further comprises:

[0204] (d) a fourth container and reagents required for performing transcription or reverse transcription-transcription located in the fourth container.

[0205] In another preferred embodiment, the kit further comprises:

[0206] (f) a fifth container and a divalent metal ion solution located in the fifth container.

[0207] One-step detection

[0208] "One-step detection" is a simple and fast nucleic acid detection solution based on the high compatibility of room-temperature Ago such as reverse transcriptase and HpAgo with reaction temperature and reaction system, which places the reverse transcription-transcription reaction and detection reaction in a PCR reaction tube for isothermal detection. It eliminates the need for temperature change, centrifugation, pipetting, tube replacement and other operations, greatly simplifies the detection device and process, and greatly reduces the possibility of contamination when opening the cover. It is conducive to automated large-scale sample processing and easy to popularize the application of home molecular diagnosis.

[0209] The main advantages of the present invention include:

[0210] (1) The present invention has developed for the first time a nuclease targeting target RNA with broad application prospects. Specifically, the present invention obtains the nuclease HpAgo by in vitro expression and purification and separation, and obtains its optimal reaction parameters through a large number of experiments. The experimental results show that the optimal reaction temperature of the nuclease HpAgo of the present invention is between 20°C and 37°C; the concentration of divalent metal ions in the range of 500mM to 3M can significantly promote the cleavage activity of HpAgo, and the present invention also unexpectedly found that the nuclease HpAgo of the present invention is intolerant to NaCl. In addition, the reaction system of the present invention has greatly improved efficiency after optimization, and the target belongs to a type of RNA that is currently less discovered, which is expected to provide new enzyme resources for in vivo RNA editing and nucleic acid detection.

[0211] (2) The present invention provides for the first time the characterization and application of a novel room-temperature Argonaute protein. Specifically, the present invention provides a nucleic acid cleavage system, characterized in that the nucleic acid cleavage system comprises: (a) guide DNA / RNA (gDNA / gRNA); (b) programmable endonuclease Argonaute (Ago); and (c) an optional reporter nucleic acid, wherein if the reporter nucleic acid is cleaved, the cleavage can be detected. The programmable endonuclease HpAgo of the present invention has an efficient and relatively complete nucleic acid cleavage function and has a strong potential for gene manipulation.

[0212] (3) The present invention breaks through the limitations of traditional nucleic acid RNA detection methods in terms of time, cost and operational difficulty, and establishes a nucleic acid detection method with high sensitivity, good specificity, rapidity and high efficiency.

[0213] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0214] Unless otherwise specified, the reagents and materials used in the examples of the present invention are all commercially available products.

[0215] Example 1 Obtaining the HpAgo gene sequence

[0216] The archaeal halophilic eutrophic bacteria (WP_058572808.1) were selected and multiple sequence alignment analysis was performed with known prokaryotic Agos using MEGA software, and a phylogenetic tree was constructed ( Figure 1). The gene sequence was synthesized after codon optimization and cloned into the pET28a expression vector.

[0217] Example 2: Heterologous expression and purification of HpAgo protein

[0218] The above-mentioned HpAgo-pET28a prokaryotic expression plasmid was introduced into E.coli BL21 (DE3) (purchased from Shanghai Shenggong) to obtain the HpAgo-pET28a / E.col i BL21 (DE3) prokaryotic expression strain. The expression strain E.coliBL21 (DE3) containing the recombinant plasmid HpAgo-pET28a was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C, 220rpm shaking to OD600 between 0.6-0.8, and IPTG was added at a final concentration of 0.4-0.6mM, and the culture was continued for 16-20h at 18°C, 200rpm shaking to induce the expression of HpAgo protein. The cells were collected by centrifugation, and the cells were resuspended in a resuspension buffer (containing 20mM Tris-HCl, pH 8.0, 1MNaCl), and then the cells were crushed by high pressure and centrifuged to obtain the supernatant. The protein was affinity purified using Ni-NTA columns (purchased from GE), 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 20mM Tris-HCl (Shanghai Bioengineering), and the protein was determined by a BCA kit (Shanghai Yisheng), and the determination steps were carried out according to the operating instructions. BSA was used as a standard, a standard solution was prepared, and a standard curve was drawn. The concentration of the purified target protein was calculated based on this, and the protein was stored in a -80℃ refrigerator for later use. HpAgo protein was analyzed by SDS-PAGE electrophoresis.

[0219] The results are as follows Figure 2 The results showed that the target protein HpAgo could be expressed in a soluble form, and the purity of the target protein was greater than 85%, which could be used for subsequent characterization analysis.

[0220] Example 3: Comparison of HpAgo with other known Ago sequences

[0221] In this example, the MegaX software was used to perform multiple sequence alignment of HpAgo with some characterized Ago to identify its conserved active center. The results showed that HpAgo has a large number of amino acids and a large protein molecular weight. The study found that the targeted cleavage of all catalytically active Ago proteins is mediated by a conserved DEDX (X represents histidine, aspartic acid or asparagine) quadruplex. By sequence alignment, it can be found that the DEDD quadruplex ( Figure 3 ). Therefore, it is further speculated that it may have nuclease catalytic activity, which requires further in vitro identification and characterization.

[0222] Example 4: HpAgo cleavage activity assay

[0223] Design of fluorescently modified 45nt single-stranded DNA, RNA target nucleic acid and four complementary 16nt DNA ( Figure 4 ), RNA guide chain, and sent it to the company for synthesis.

[0224] DNA target nucleic acid sequence:

[0225] 5'-FAM-CGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGG(SEQ ID NO:2)-3'

[0226] RNA target nucleic acid:

[0227] 5'-FAM-CGCAGCAUGUCAAGAUCACAGAUUUUGGGCUGGCCAAACUGCUGG(SEQ ID NO:3)-3'

[0228] gDNA:

[0229] 5'-HO / P-TAGTTTGGCCAGCCCA(SEQ ID NO:4)-3'

[0230] gRNA:

[0231] 5'-HO / P-UAGUUUGGCCAGCCCA(SEQ ID NO:5)-3'

[0232] Prepare a reaction buffer (containing 15mM Tris-HCl pH8.0, 250mM NaCl), add 0.5mM MnCl2, 200nM HpAgo, 2μM synthetic gDNA or gRNA and 0.8μM 5'fluorescently modified sequence-complementary single-stranded DNA or RNA target nucleic acid to the reaction buffer, react at 80°C for 30min, after the reaction, take 6-10μL sample, add loading buffer (containing 95% (deionized) formamide, 0.5mmol / L EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol) at a ratio of 1:1, and perform electrophoresis detection under 16% nucleic acid Urea-PAGE.

[0233] The results are as follows Figure 5 The results showed that HpAgo can use 5'P-gDNA and 5'P-gRNA to cleave complementary single-stranded RNA.

[0234] Example 5: Analysis of HpAgo catalytic properties

[0235] 8-35nt 5'phosphorylated gDNA (Table 1) and 5'phosphorylated gRNA (Table 2) were designed respectively to explore the effect of different lengths of gDNA / gRNA on HpAgo enzyme activity. In the reaction buffer (15mM Tris-HCl pH8.0, 250mM NaCl) of Example 4, a final concentration of 0.5mM MnCl2, a final concentration of 800nM HpAgo, 800nM synthesized gDNA / gRNA of different lengths and 200nM 45nt sequence complementary single-stranded DNA target nucleic acid were added, and the reaction was carried out at 25°C for 60min, and the reaction products were detected by electrophoresis under 16% nucleic acid Urea-PAGE.

[0236] The results are as follows Fig.11 The results showed that HpAgo can use 5'-P gDNA with a length of 8-30nt, and can also use 5'-P gRNA with a length of 8-30nt to cleave complementary target nucleic acids ( Fig.11 ).

[0237] Table 1

[0238]

[0239]

[0240] Table 2

[0241]

[0242] The enzymatic activity of HpAgo was investigated at different temperatures (10°C, 20°C, 25°C, 37°C, 45°C, 50°C, 55°C), and MnCl2 with a final concentration of 0.5 mM, HpAgo with a final concentration of 800 nM, 800 nM synthetic gDNA / gRNA and 200 nM 45 nt sequence complementary single-stranded RNA target nucleic acid were added to the reaction buffer of Example 4 (15 mM Tris-HCl pH8.0, 250 mM NaCl). The reaction was carried out at different temperatures for 60 min, and the reaction products were detected by electrophoresis under 16% nucleic acid Urea-PAGE.

[0243] The results are as follows Figure 6 The results showed that HpAgo can cleave complementary target nucleic acids using 5'-P gDNA / 5'-PgRNA in the range of 10°C-45°C.

[0244] The concentrations of HpAgo, guide strand and target nucleic acid remained unchanged, and CoCl2, CuCl2, MgCl2, MnCl2, ZnCl2 and CaCl2 solutions with a final concentration of 0.5 mM were added to the reaction system, respectively. The reaction temperature was 25°C, and the reaction was carried out for 60 min. 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, and different final concentrations of MnCl2 or CoCl2 were added: 20 μM, 50 μM, 100 μM, 250 μM, 500 μM, 750 μM, 1000 μM, 1500 μM, 2000 μM, 3000 μM, 5000 μM, to determine the optimal MnCl2 or CoCl2 concentration of HpAgo mediated by the 5' phosphorylated guide strand.

[0245] The results are as follows Figure 7 , Figure 8 and Fig. 9 The results showed that HpAgo can utilize Mn 2+ and Co 2+ As a metal ion, it mediates single-stranded DNA / RNA-guided RNA shearing. Among them, HpAgo prefers Mn 2+ , and 1000μM-5000μM Mn 2+ It can keep HpAgo highly active, 1000uM-1500uM Co 2+ It can keep HpAgo highly active.

[0246] The reaction buffer components were adjusted to prepare reaction buffers with a final concentration of 15 mM Tris-HCl pH 8.0 and different concentrations of NaCl (50 mM, 100 mM, 250 mM, 500 mM, 1000 mM, 1500 mM, 2000 mM) (prepared with ultrapure water). Other reaction systems remained unchanged. The reaction was carried out at 25° C. for 60 min, and the electrophoresis was performed under 16% nucleic acid Urea-PAGE.

[0247] The results are as follows Fig.10 The results showed that HpAgo could exert higher cleavage activity when the NaCl concentration was 0-250 mM.

[0248] 16nt gDNA / gRNA with A, T, G, and C as the first base at the 5' end were designed (Tables 3 and 4), the reaction system remained unchanged, MnCl2, gDNA / gRNA, and target RNA were added, and the preference of HpAgo for the first base at the 5' end of gDNA / gRNA mediated by the 5' phosphorylated guide strand was determined. The reaction was carried out at 25°C for 60 minutes, and electrophoresis was performed under 16% nucleic acid Urea-PAGE.

[0249] Table 3

[0250]

[0251] Table 4

[0252]

[0253] The results are as follows Fig.12 The results show that HpAgo has a preference for the absence of the first base at the 5' end of gDNA / gRNA.

[0254] Studying the relationship between guide and target mismatch and Ago activity is of great significance for its application of substrate specificity in nucleic acid detection to accurately distinguish substrates. A series of gDNA / gRNA with single-point mismatches at different sites (0-16) with the target were designed. The reaction system remained unchanged, MnCl2, gDNA / gRNA with different site mismatches and target RNA were added to measure the distinguishing shearing effect of HpAgo. The reaction was carried out at 25℃ for 60 minutes, and electrophoresis was performed under 16% nucleic acid Urea-PAGE. The specific sequence information of single-point mismatch is shown in Table 5-Table 6, and the specific sequence information of double mismatch is shown in Table 7-Table 8.

[0255] Table 5

[0256]

[0257]

[0258] Table 6

[0259]

[0260] Table 7

[0261]

[0262]

[0263] Table 8

[0264]

[0265] The results are as follows Fig.13 The results show that when a single mismatch occurs at positions 2-5 of gDNA or gRNA, the cleavage efficiency of HpAgo is significantly reduced. Fig.14 As shown, when double mismatches occurred at positions 2-8 and 12-16 of gDNA or gRNA, the cleavage efficiency of HpAgo was significantly reduced.

[0266] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings 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 claims attached to this application.

Claims

1. A detection system for directly detecting a target nucleic acid molecule, the system comprising: (a) a guide gDNA pair or a guide gRNA pair; (b) a room-temperature Argonaute protein (Ago enzyme), wherein the room-temperature Argonaute protein (Ago enzyme) is a room-temperature Argonaute protein (Ago enzyme) that uses DNA or RNA as a guide to guide the cleavage of a target RNA; (c) a fluorescent reporter nucleic acid having a fluorescent group and a quencher group; in, The target nucleic acid molecule is a target DNA or a target RNA.

2. The detection system according to claim 1, characterized in that In the detection system, the target DNA is transcribed to form a target RNA, which is recognized and sheared by the room temperature Argonaute protein (Ago enzyme).

3. The detection system according to claim 1, characterized in that In the detection system, the target RNA is reverse transcribed and transcribed to form a target RNA, which is recognized and sheared by the room temperature Argonaute protein (Ago enzyme).

4. The detection system according to claim 1, characterized in that The room temperature Argonaute protein (Ago enzyme) has the ability to guide the cleavage of target RNA using DNA or RNA as a guide.

5. The detection system according to claim 1, characterized in that The normal temperature Argonaute protein (Ago enzyme) is derived from normal temperature halophilic bacteria Haloferax_profundi, Haloferax larsenii, Haloferax alexandrinus, Haloferax alexandrinus, Halocatena pleomorpha, Halorussus marinus, Halorubrum sp.48-1-W, Haloarchaeobius salinus, Halosimplex salinum, Natronomonasgomsonensis, Halogeometricum borinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculus aciditolerans, Halalkaliarchaeumdesulfuricum, Halosimplex pelagicum.

6. The detection system according to claim 1, characterized in that The normal temperature Argonaute protein (Ago enzyme) is selected from the following group (Haloferax_profundi) (HpAgo), Haloferax larsenii, Haloferax alexandrinus (HaleAgo), Haloferax alexandrinus (HaalAgo), Halocatena pleomorpha (HaplAgo), Halorussus marinus (HamaAgo), Halorubrum sp.48-1-W (HaspAgo enzyme), Haloarchaeobiussalinus (HalosaAgo), Halosimplex salinum, Natronomonas gomsonensis, Halogeometricum borinquense, Halogeometricum limi, Halosolutus gelatinilyticus, Halorussus, Halocalculus aciditolerans, Halalkaliarchaeum desulfuricum, Halosimplex pelagicum and its mutants.

7. The detection system according to claim 1, characterized in that The amino acid sequence of the room temperature Argonaute protein (Ago enzyme) is shown in SEQ ID NO.

1.

8. The detection system according to claim 1, characterized in that The working temperature of the room temperature Argonaute protein (Ago enzyme) is 5-60°C, preferably 10-45°C, and more preferably 25-37°C.

9. A kit for directly detecting a target nucleic acid molecule, characterized in that: The kit comprises: (i) the detection system according to claim 1 or a reagent for preparing the detection system; and (ii) instructions for use, wherein the instructions describe a method for directly detecting a target nucleic acid molecule using the detection system.

10. A method for directly detecting whether a target nucleic acid molecule exists in a sample, characterized in that: The following steps are involved: (a) providing the detection system for detecting target nucleic acid molecules according to claim 1; and (b) reacting the detection system with a sample to be detected at a certain temperature to form a first reaction solution; (c) performing fluorescence detection on the first reaction solution to obtain a fluorescence signal value; Among them, if a fluorescence signal value is detected in the first reaction solution, it means that a target nucleic acid molecule exists in the sample; and if no fluorescence signal value is detected in the first reaction solution, it means that no target nucleic acid molecule exists in the sample, and the target nucleic acid molecule is a target DNA or a target RNA.

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