WO2022222973A1 - 一种新型中温原核Argonaute蛋白PbAgo表征及应用 - Google Patents

一种新型中温原核Argonaute蛋白PbAgo表征及应用 Download PDF

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WO2022222973A1
WO2022222973A1 PCT/CN2022/088007 CN2022088007W WO2022222973A1 WO 2022222973 A1 WO2022222973 A1 WO 2022222973A1 CN 2022088007 W CN2022088007 W CN 2022088007W WO 2022222973 A1 WO2022222973 A1 WO 2022222973A1
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detection system
pbago
nucleic acid
molecule
amino acid
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French (fr)
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冯雁
董华蓉
黄飞
许晓忆
李忠磊
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上海交通大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to the characterization and application of a novel mesophilic prokaryotic Argonaute protein PbAgo.
  • the purpose of the present invention is to provide a rapid, cheap and efficient small molecule detection technology.
  • the present invention specifically provides a small molecule detection method based on the mesophilic prokaryotic Argonaute protein PbAgo.
  • a detection system for detecting a target molecule comprising:
  • the target molecules include nucleic acid molecules and small chemical molecules.
  • the target molecule is a nucleic acid molecule, and the nucleic acid molecule can be the same molecule as the reporter nucleic acid.
  • the nucleic acid molecule is in a supercoiled state.
  • the GC content in the nucleic acid molecule is ⁇ 36%, preferably ⁇ 20%, more preferably ⁇ 14%.
  • the detection system further includes: (d) an allosteric transcription factor, the allosteric transcription factor has both binding to the reporter nucleic acid and The activity of the small chemical molecule, and the binding of the reporter nucleic acid and the small chemical molecule to the allosteric transcription factor is competitive.
  • the allosteric transcription factor is selected from the group consisting of HosA, TetR, HucR and the like.
  • the small chemical molecule is selected from the group consisting of p-hydroxybenzoic acid (p-HBA), tetracycline, uric acid and the like.
  • the allosteric transcription factor is HosA
  • the small chemical molecule is p-hydroxybenzoic acid (p-HBA).
  • the nuclease Argonaute is derived from Paenibacillus borealis, Clostridium butyricum, Clostridium perfringens, Limnothrox rosea), Intestinibacter bartlettii, Natronobacterium gregoryi, Pyrococcus furiosus, or Thermus thermophiles.
  • the nuclease Argonaute is derived from Paenibacillus borealis, and the nuclease Argonaute is the nuclease PbAgo.
  • the PbAgo includes wild-type and mutant-type PbAgo.
  • the nucleic acid enzyme PbAgo comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the working temperature of the nuclease is 10-65°C, preferably 20-55°C, more preferably 30-45°C, more preferably 30-37°C.
  • the guide DNA is a single-stranded DNA molecule phosphorylated or hydroxylated at the 5' end.
  • the guide DNA is a single-stranded DNA molecule phosphorylated at the 5' end.
  • the guide DNA and the reporter nucleic acid have complementary fragments.
  • the length of the guide DNA is 8-35nt, preferably 14-21nt, and most preferably 15-18nt.
  • the first nucleotide at the 5' end of the guide DNA is a phosphorylated or hydroxylated thymine (T).
  • nucleotide sequence of the guide DNA is shown in SEQ ID NO:3.
  • the reporter nucleic acid is single-stranded DNA (ssDNA).
  • the cleavage when the reporter nucleic acid is cleaved, the cleavage can be detected by electrophoresis.
  • the reporter nucleic acid is a fluorescent reporter nucleic acid
  • the fluorescent reporter nucleic acid has a fluorescent group and/or a quenching group.
  • the fluorescent group and the quenching group are independently located at the 5' end and the 3' end of the fluorescent reporter nucleic acid.
  • 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.
  • the reporter nucleic acid is single-stranded DNA (ssDNA) with a length of 10-100nt, preferably 20-70nt, more preferably 30-60nt, more preferably 40-50nt, most preferably 45nt.
  • ssDNA single-stranded DNA
  • the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof.
  • the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.
  • the fluorescent group is FAM.
  • the detection system further includes: (e) divalent metal ions.
  • the divalent metal ions are 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.
  • the divalent metal ions are selected from the group consisting of Mg 2+ , Mn 2+ , or a combination thereof.
  • the concentration of divalent metal ions is 10mM-3M, preferably 500mM-3M, more preferably 1M-3M.
  • the detection system further comprises: (f) a buffer.
  • the concentration of NaCl is ⁇ 750 mM, preferably ⁇ 500 mM, more preferably ⁇ 100 mM.
  • the pH value of the buffer solution is 7-9, preferably 8.0.
  • the detection system further contains the target molecule to be detected.
  • the PbAgo enzyme is guided to cut the reporter nucleic acid, thereby generating a detectable signal (eg, fluorescence).
  • the concentration of the target molecule to be detected in the detection system is 0.01-10 ⁇ M, preferably 0.05-1 ⁇ M, more preferably 0.1 ⁇ M.
  • the concentration of the reporter nucleic acid is 0.01-10 ⁇ M, preferably 0.05-1 ⁇ M, more preferably 0.1 ⁇ M.
  • the concentration of the nuclease Ago is 0.5-8 ⁇ M, preferably 2-4 ⁇ M, more preferably 3 ⁇ M.
  • the concentration of the guide DNA is 0.025-4 ⁇ M, preferably 0.1-1 ⁇ M, more preferably 0.5 ⁇ M.
  • the molar ratio of the reporter nucleic acid to the target molecule is 1:1.8 to 1:72000, preferably 1:36000.
  • the molar ratio of the target molecule to be detected, the guide DNA, and the nuclease Ago is 1:(0.25-20):(1.5-120), preferably 1: (1-10):(5-50), optimally 1:5:30.
  • kit for detecting target nucleic acid molecules comprising:
  • the kit includes:
  • the kit also contains:
  • the kit also contains:
  • the kit also includes:
  • 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.
  • a method for detecting whether a target molecule exists in a sample comprising the following steps:
  • the shear signal value is detected in the reaction solution, it indicates that the target molecule exists in the sample; and if the shear signal value is not detected in the reaction solution, it indicates that the target molecule does not exist in the sample.
  • the detection includes qualitative detection and quantitative detection.
  • the detection in step (c) may include: using electrophoresis to identify the length of the reporter nucleic acid, so as to determine whether the reporter nucleic acid is cleaved.
  • the detection in step (c) includes: using a microplate reader or a fluorescence spectrophotometer for detection.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • nuclease Argonaute is provided for preparing a reagent or kit for detecting target molecules.
  • nuclease Argonaute is derived from Paenibacillus borealis; or a homologous analog thereof with the same or similar functions.
  • the PbAgo includes wild-type and mutant-type PbAgo.
  • nucleic acid enzyme Argonaute has an amino acid sequence selected from the group consisting of:
  • amino acid residues On the basis of the sequence shown in SEQ ID NO: 1, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 10 amino acid residues are added at its N-terminus or C-terminus (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 and the sequence shown in SEQ ID NO: 1 have ⁇ 85 % (preferably ⁇ 90%, more preferably ⁇ 95%, such as ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%) sequence identity; and the amino acid sequence obtained is identical to (i) or similar function.
  • the present invention first provides a gene encoding the mesophilic prokaryotic Argonaute protein PbAgo.
  • the Ago protein with high sequence consistency and derived from the mesophilic host was selected, and then the whole gene data of the host Paenibacillus borealis strain DSM 13188 was mined to obtain the full-length gene. 2118bp, encoding 705 amino acids.
  • PbAgo contains the DEDX catalytic residue of Ago protein that exerts splicing activity.
  • the present invention constructs a recombinant plasmid pET-28a(+)-TEV-PbAgo, the recombinant plasmid is transformed into Escherichia coli BL21(DE3), and the expression is induced by IPTG at low temperature, thereby realizing the heterologous active expression of PbAgo, and the supernatant after the cell is broken
  • SDS-PAGE results showed a protein band with higher concentration, and the molecular weight was consistent with the molecular weight of the target protein, indicating that PbAgo can produce efficient soluble expression in E. coli.
  • the molecular weight of the novel mesophilic prokaryotic Argonaute protein PbAgo obtained by the present invention is about 80.79kDa, and under the guidance of 5'P gDNA, the optimum reaction temperature is between 30°C and 55°C; under the guidance of 5'OH gDNA, the optimum reaction temperature is The reaction temperature is between 55°C and 65°C.
  • Divalent metal ions such as Mn 2+ and Mg 2+ can promote the shearing activity of PbAgo, and when the concentration of divalent metal ions is in the range of 500mM to 3M, the PbAgo enzyme activity maintains a high level.
  • the enzyme is intolerant to NaCl, which inhibits the cleavage of ssDNA by PbAgo. After measuring its cleavage kinetics, it can be seen that under the guidance of 5'P DNA guide, PbAgo reacts very quickly, and can cleave 95% of the substrate within 30min. It will have broad prospects in gene editing in living
  • the PbAgo obtained by the present invention can bind to gDNA at 30° C. and cut irregular double-stranded DNA.
  • the allosteric transcription factor HosA can bind to irregular dsDNA.
  • the small molecule compound p-HBA exists, it competes for binding to HosA, and the released irregular dsDNA can be cleaved by PbAgo. This property of PbAgo can be combined with HosA for the detection of small molecule compounds.
  • Figure 1 shows the results of multiple sequence alignment of PbAgo and reported prokaryotic Ago. It can be seen from the figure that PbAgo, like other reported prokaryotic Agos, also contains a DEDX active center that exerts cleavage activity.
  • Figure 2 shows the results of SDS-PAGE electrophoresis of recombinant strains expressing PbAgo.
  • M is a protein marker
  • 1 is a purified solution of PbAgo.
  • Figure 3 shows the designed synthetic 45nt ssDNA/ssRNA target sequence (target) and its complementary paired 21nt 5'P/OH modified DNA/RNA guide sequence (guide).
  • Figure 4 shows the preference of purified PbAgo for guide and target sequences. It can be seen from the figure that PbAgo can use 5'P/OH gDNA to cleave ssDNA complementary to gDNA at 37 °C.
  • Figure 5 shows the optimal temperature for PbAgo to exert shearing activity under the guidance of two different gDNAs.
  • the optimum reaction temperature is between 30°C-55°C; under the guidance of 5'OH gDNA, the optimum reaction temperature is between 55°C-65°C.
  • Figure 6 shows the preference of PbAgo for divalent metal ions under the guidance of two different gDNAs. It can be seen from the figure that divalent metal ions such as Mn 2+ and Mg 2+ can promote the shear activity of PbAgo.
  • Figure 7 shows the effect of different divalent metal ion (Mn 2+ ) concentrations on the cleavage activity of PbAgo under the guidance of two different gDNAs. It can be seen from the figure that the PbAgo enzyme activity maintains a high level when the divalent metal ion concentration is in the range of 500mM to 3M.
  • Figure 8 shows the tolerance of PbAgo to NaCl under the guidance of two different gDNAs. It can be seen from the figure that the enzyme is intolerant to NaCl, and NaCl can inhibit the activity of PbAgo to cut ssDNA.
  • Figure 9 shows the cleavage kinetics of two different gDNA-directed PbAgos determined under optimal reaction conditions.
  • Figure 10 shows the shear kinetics of two different gDNA-directed PbAgos in the first 20 min determined under optimal reaction conditions. It can be seen from the figure that PbAgo has higher shear rate and shear efficiency under the guidance of 5'P gDNA.
  • Figure 11 shows the effect of gDNA length on PbAgo cleavage activity. It can be seen from the figure that when the length of gDNA is 13-30nt, PbAgo has shearing activity; when the length of gDNA is 14-21nt, the shearing efficiency of PbAgo is higher; when the length of gDNA is 15-18nt, the shearing efficiency of PbAgo is the best.
  • Figure 12 shows the results of the detection of the small molecule compound p-HBA by PbAgo.
  • PbAgo/gDNA still competes with HosA for binding to dsDNA in sample No. 4 without adding p-HBA.
  • sample No. 5 adding p-HBA the difference is small, resulting in the detection of high background value. It can be seen that HosA and PbAgo interfere with each other due to the common substrate, and corresponding adjustments can be made to this problem in the future.
  • Figure 13 shows the result of shearing plasmid pUC19 by PbAgo under the guidance of a pair of 5'P gDNA at 37°C for 3h. It can be seen from the figure that in sample No. 7, PbAgo can use a pair of 5'P gDNA to target one strand of the plasmid respectively, and shear the supercoiled plasmid to generate linearized plasmid DNA.
  • OC open-circle plasmid (one strand of plasmid is broken); LIN: linearized plasmid (double strand of plasmid is broken); SC: supercoiled plasmid.
  • Figure 14 shows the result of shearing the plasmid pUC19 by PbAgo under the guidance of a pair of 5'OH gDNA at 65°C for 3h. It can be seen from the figure that in sample No. 7, PbAgo can use a pair of 5'OH gDNA to target one strand of the plasmid respectively, and shear the supercoiled plasmid to generate linearized plasmid DNA.
  • Figure 15 shows the effect of plasmid target fragment GC content on PbAgo cleavage efficiency.
  • plasmid pUC19 6 target fragments with different GC contents of 50bp were found, and 6 pairs of 5'P gDNA complementary to them were designed and synthesized. It can be seen from the figure that the lower the GC content of the 50bp target DNA fragment, the better the shearing effect of PbAgo.
  • PbAgo can cut double-stranded DNA fragments with a GC content of not more than 36% to generate linearized plasmids; however, although the GC content of the fragment is as high as 70%, PbAgo can make part of pUC19 open-loop state, which may be due to the fact that PbAgo is in the 5'P gDNA One strand of the plasmid was cut under guidance.
  • the inventors developed a target molecule detection method based on a novel nuclease Ago for the first time.
  • the present invention obtained the nuclease PbAgo through in vitro expression, purification and separation, and obtained its optimal reaction parameters through a large number of groping experiments.
  • the experimental results show that the optimum reaction temperature of the nuclease PbAgo of the present invention is between 30°C and 65°C; divalent metal ions such as Mn 2+ , Mg 2+ , and the concentration of divalent metal ions can be significantly in the range of 500mM to 3M.
  • the nuclease of the present invention is intolerant to NaCl.
  • the PbAgo of the present invention can react rapidly at 30°C, which can cleave 95% of the substrate (ie, irregular dsDNA) within 30 min.
  • the reaction system of the present invention can also be used to cut plasmid DNA in a supercoiled state, which is expected to provide new enzyme resources for gene editing in vivo. The present invention has been completed on this basis.
  • 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 refers 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.
  • 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 .
  • the present invention also provides non-conservative amino acid substitutions that allow for amino acid substitutions from different groups.
  • 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.
  • PIWI P element-induced wimpy testis
  • RNA interference RNA interference
  • eAgos Eukaryotic Argonaute protein
  • RISC multi-protein RNA-induced silencing complex
  • 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.
  • 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.
  • pAgos prokaryotic Ago
  • nuclease Paenibacillus borealis As used herein, the terms “nuclease Paenibacillus borealis”, “nuclease Paenibacillus borealis”, “PbAgo enzyme” are used interchangeably and refer to the nucleases described in the detection system of the first aspect of the present invention.
  • the wild-type PbAgo enzyme has the amino acid sequence shown in SEQ ID NO:1.
  • the PbAgo enzymes of the present invention may also comprise mutant forms thereof that retain functional activity.
  • the mutant form can contain one or more amino acid residue substitutions, deletions, changes or insertions on the basis of the sequence shown in SEQ ID NO: 1, or add 1 to 10 to its N-terminus or C-terminus.
  • amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues), thereby obtaining an amino acid sequence; and the obtained amino acid sequence is the same as shown in SEQ ID NO: 1 the sequence has ⁇ 85% (preferably ⁇ 90%, more preferably ⁇ 95%, such as ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%) sequence identity; and the amino acid sequence obtained has the same Type PbAgo enzymes have the same or similar functions.
  • 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 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 suitable for the functional activity of the Ago enzyme (especially the PbAgo enzyme) of the present invention.
  • a detection system for detecting a target molecule comprising: (a) guide DNA (gDNA); (b) nuclease Argonaute (Ago); and (c) reporter nucleic acid, wherein, if the reporter nucleic acid is cleaved, the cleavage can be detected; wherein, the target nucleic acid molecule is target DNA.
  • the working temperature of the nuclease is 10-65°C, preferably 20-55°C, more preferably 30-45°C, more preferably 30-37°C .
  • the guide DNA is a single-stranded DNA molecule phosphorylated or hydroxylated at the 5' end; more preferably, the guide DNA is a single-stranded DNA molecule phosphorylated at the 5' end.
  • the length of the guide DNA is 8-35nt, preferably 14-21nt, and most preferably 15-18nt.
  • the reporter nucleic acid is single-stranded DNA (ssDNA).
  • ssDNA single-stranded DNA
  • the reporter nucleic acid when the reporter nucleic acid is cleaved, the cleavage can be detected by electrophoresis.
  • the reporter nucleic acid is a fluorescent reporter nucleic acid bearing a fluorophore and a quencher group.
  • the fluorescent group and the quenching group are independently located at the 5' end and the 3' end of the fluorescent reporter nucleic acid; preferably, the fluorescent group and the quenching group are respectively located in the fluorescent reporter nucleic acid. flanked by the complementary regions of the guide DNA.
  • the fluorescent group includes: FAM, HEX, CY5, CY3, VIC, JOE, TET, 5-TAMRA, ROX, Texas Red-X, or a combination thereof.
  • the quenching group includes: BHQ, TAMRA, DABCYL, DDQ, or a combination thereof.
  • the length of the reporter nucleic acid is 10-100nt, preferably 20-70nt, more preferably 30-60nt, more preferably 40-50nt, and most preferably 45nt.
  • the reporter nucleic acid in the detection system of the present invention can also be the same molecule as the target molecule.
  • the nucleic acid molecule is plasmid DNA in a supercoiled state. More preferably, the GC content in the nucleic acid molecule is no more than 36%, preferably no more than 20%, more preferably no more than 14%.
  • Divalent metal ions may also be included in the detection system of the present invention.
  • the divalent metal ions are selected from the group consisting of Mg 2+ , Mn 2+ , Fe 2+ , Co 2+ , Cu 2+ , Ni 2+ , Zn 2+ , Ca 2+ , or a combination thereof. Preferred are: Mg 2+ , Mn 2+ , or a combination thereof.
  • the concentration of divalent metal ions is 10mM-3M, preferably 500mM-3M, more preferably 1M-3M.
  • the concentration of NaCl is ⁇ 750 mM, preferably ⁇ 500 mM, more preferably ⁇ 100 mM.
  • the pH value of the detection system of the present invention is 7-9, preferably 8.0.
  • the terms "detection method of the present invention” and “nucleic acid detection method based on Argonaute protein” are used interchangeably and refer to the detection method described in the third aspect of the present invention.
  • a method for detecting whether a target molecule exists in a sample comprising the following steps: (a) providing the detection system for detecting the target molecule according to the present invention; and (b) applying the detection system to the React with the sample to be detected at a certain temperature to form a reaction solution; (c) detect the reaction solution to obtain a shear signal value; wherein, if the shear signal value is detected in the reaction solution, then It means that the target molecule exists in the sample; and no shear signal value is detected in the reaction solution, it means that the target molecule does not exist in the sample.
  • the detection includes qualitative detection and quantitative detection.
  • the detection in step (c) may include: using electrophoresis to identify the length of the reporter nucleic acid, so as to determine whether the reporter nucleic acid is cleaved. If a shorter length nucleic acid product is produced, this indicates that the reporter nucleic acid is cleaved by Ago in the detection system.
  • the detection in step (c) includes: using a microplate reader or fluorescence spectrophotometry meter for detection.
  • the method is an in vitro method. In another embodiment, the method is non-diagnostic and non-therapeutic.
  • kits for detecting target nucleic acid molecules comprising: (i) the detection system of the present invention or a reagent for preparing the detection system; and (ii) instructions for use, the instructions Methods for the detection of target molecules using the described detection system are described.
  • the kit comprises: (a) a first container and a guide DNA located in the first container; (b) a second container and a nuclease Argonaute (Ago) located in the second container; and (c) a third container and a reporter nucleic acid located in the third container.
  • the kit further contains: (d) a fourth container and an allosteric transcription factor located in the fourth container.
  • the kit further contains: (e) a fifth container and a divalent metal ion located in the fifth container.
  • the containers described above may be the same or different containers.
  • the PbAgo in the detection system of the present invention can use two kinds of gDNA with different 5' end modifications (5'P/OH) to cut ssDNA.
  • PbAgo in the detection system of the present invention has a fast reaction rate of shearing ssDNA under the guidance of 5'P gDNA, and the reaction time is short, and it only takes 30 minutes to achieve nearly 100% shearing.
  • PbAgo in the detection system of the present invention has high tolerance to NaCl, and when the NaCl concentration in the reaction system is not higher than 750 mM, PbAgo has higher shearing activity.
  • PbAgo in the detection system of the present invention has lower requirements on the length of gDNA, and when the gDNA is 14-21 nt, PbAgo has higher shearing activity.
  • the PbAgo in the detection system of the present invention can shear the plasmid dsNDA (pUC19) with a GC content not higher than 36% under the guidance of a pair of 5'P/OH gDNA.
  • the present invention uses Ago and allosteric transcription factor together to detect small molecule compounds for the first time.
  • PbAgo can utilize the allosteric effect of HosA to detect p-HBA with a detection rate much higher than other detection methods known in the art.
  • the amino acid sequence similarity of CbAgo was searched by BLAST in the NCBI database (https://www.ncbi.nlm.nih.gov/), and some parts of the sequence were selected with high consistency and were derived from Agos of mesophilic hosts.
  • the amino acid sequence and the reported prokaryotic Agos were analyzed with MEGA X software to construct a homologous evolutionary tree, and the PbAgo that was closely related to the reported mesophilic prokaryotic Agos was selected. It was then determined by multiple sequence alignment analysis with the reported prokaryotic Agos whether it contained DEDX catalytic residues.
  • the gene sequence of PbAgo was constructed into pET-28a(+)-TEV vector to synthesize plasmid.
  • Escherichia coli BL21 (DE3) was transformed with the synthesized recombinant plasmid, and then single-clonal transfer test tubes were picked from the transformed plates for culture, and the glycerol bacteria were preserved.
  • Figure 2 shows the results of SDS-PAGE electrophoresis of recombinant strains expressing PbAgo.
  • the bacterial liquid containing the pET-28a(+)-TEV-PbAgo expression vector was inoculated into 5mL LB liquid medium (containing 50 ⁇ g/mL kanamycin), and cultured overnight at 220rpm and 37°C; transfer at 1% inoculum amount To 1L LB liquid medium (containing 50 ⁇ g/mL kanamycin), culture at 220rpm, 37°C until the OD 600 reaches 0.6-0.8, add IPTG with a final concentration of 0.5mM, induce expression at 16°C, 220rpm for 16h-18h .
  • the cells were collected by centrifugation at 6000 rpm and 4°C for 30 min, and the cells were resuspended in resuspension buffer (containing 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 2% glycerol), and then high-pressured with a pre-cooled homogenizer. broken. The supernatant was obtained by centrifuging at 10,000 rpm and 4°C for 30 min, and then purified by Ni-NTA column to obtain the purified solution of PbAgo, which was detected and identified by SDS-PAGE.
  • resuspension buffer containing 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 2% glycerol
  • the purified target protein was concentrated to 2.5 mL with an ultrafiltration tube at 4°C and 4500 rpm, and then further purified to remove imidazole.
  • the enzyme concentration was determined by the BCA kit, and the determination steps were carried out according to the operating instructions. Using BSA as a standard, prepare a standard solution, draw a standard curve, calculate the concentration of the purified target protein, and store the target protein at -80°C.
  • Fluorescently modified ssDNA and ssRNA target nucleic acids and four complementary gDNA and gRNA were designed and sent to GenScript Biotechnology Co., Ltd. for synthesis.
  • PbAgo (3 ⁇ M) and ssDNA or ssRNA guide (0.5 ⁇ M) were added to the reaction buffer (15 mM Tris-HCl, pH 8.0, 200 mM NaCl, 2 mM MnCl 2 ), mixed well, and incubated at room temperature for 15 min. Then, a fluorescently labeled ssDNA or ssRNA target strand (the ratio of target:guide:Ago is 1:5:30) was added, and the reaction was carried out at 37°C for 30 min.
  • loading buffer containing 95% (deionized) formamide, 0.5mM EDTA, 0.025% bromophenol blue, 0.025% xylene blue
  • loading buffer containing 95% (deionized) formamide, 0.5mM EDTA, 0.025% bromophenol blue, 0.025% xylene blue
  • reaction system remained unchanged.
  • PbAgo and gDNA were added to the reaction buffer (15mM Tris-HCl pH8.0, 200mM NaCl, 2mM MnCl 2 ), mixed well, and incubated at room temperature for 15 minutes. Then, fluorescently labeled ssDNA or ssRNA target was added, and the reaction was carried out at 10°C, 20°C, 30°C, 37°C, 45°C, 55°C, 65°C, 75°C, 85°C, and 95°C for 30 minutes, respectively.
  • reaction products were detected by electrophoresis under 16% nucleic acid Urea-PAGE. After the gel was stained with SYBR gold, the gel image was quantitatively analyzed with the gel quantification software ImageJ and graphed by GraphPad Prism 8.
  • reaction buffer 15 mM Tris-HCl pH8.0, 200 mM NaCl
  • MgCl 2 , MnCl 2 , FeCl 2 , CoCl 2 , CuCl 2 , NiCl 2 , ZnCl 2 , and CaCl 2 were added to a final concentration of 2 mM, respectively.
  • PbAgo and gDNA mix well, and incubate at room temperature for 15min.
  • fluorescently labeled ssDNA or ssRNA target was added, and the reaction was carried out at 37 °C for 30 min. A sample without metal ions was used as a control group.
  • reaction products were detected, stained, gel quantified and graphed as described above.
  • the reaction conditions were unchanged, and different concentrations of MnCl 2 were added to the reaction system: 0 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, 1000 mM, 2000 mM, 3000 mM, and the optimal MnCl of PbAgo under the guidance of different guides was detected. 2 concentrations.
  • reaction conditions were unchanged, and different concentrations of NaCl were added to the reaction system: 50 mM, 100 mM, 250 mM, 500 mM, 750 mM, 1000 mM, 1500 mM, 2000 mM, 2500 mM, 3000 mM.
  • the concentrations of PbAgo, gDNA and ssDNA remained unchanged. Under the concentration of 50 mM NaCl and 2 mM MnCl 2 , the reaction time at 37 °C was different: 0 min, 3 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, 80 min, 100 min, 120 min, 150 min and 180 min . Electrophoretic detection was performed under 16% nucleic acid Urea-PAGE. Gel image staining and quantitative mapping were performed as described above.
  • 5'P gDNA of different lengths are designed to be paired with it.
  • reaction system The other components and reaction conditions in the reaction system were unchanged. 0.5 ⁇ M of 5'P gDNA of different lengths was added to the system, and the reaction was carried out at 37 °C for 30 min. A sample without gDNA was used as a control group. The reaction products were detected, stained, gel quantified and graphed as described above.
  • Irregular dsDNA and allosteric transcription factor HosA were firstly added to the reaction system, and incubated at 30°C for 20 min to bind them. Then the target small molecule p-HBA was added to the reaction system to induce the dissociation of dsDNA and HosA, and then the free dsDNA was detected by PbAgo.
  • Figure 12 shows the results of the detection of the small molecule compound p-HBA by PbAgo.
  • a pair of 5'P/OH gDNA complementary to the pUC19 fragment was designed and synthesized, and sent to Sangon Bioengineering Technology Co., Ltd. for synthesis.
  • FW ssDNA TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAA (SEQ ID NO: 19)
  • RV ssDNA TTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA (SEQ ID NO: 20)
  • reaction buffer 15 mM Tris-HCl, pH 8.0, 2 mM MnCl 2
  • 600ng of pUC19 plasmid was added, and the reaction was carried out at 37°C/65°C for 3 hours.
  • loading buffer at a ratio of 5:1, and use 1% agarose gel for electrophoresis detection.
  • Figures 13 and 14 show the results of cleavage of pUC19 by PbAgo under the guidance of 5'P/OH gDNA.
  • FW ssDNA TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAA (SEQ ID NO:23)
  • RV ssDNA TTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA (SEQ ID NO: 24)
  • Rv gDNA sequence 5'P-TGAAGTTTTAAATCAATCTAA 3' (SEQ ID NO:26)
  • RV ssDNA ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT (SEQ ID NO: 28)
  • Rv gDNA sequence 5'P-AAATTAAAAATGAAGTTTTAA 3' (SEQ ID NO:30)
  • FW ssDNA GATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGT (SEQ ID NO: 31)
  • RV ssDNA ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC (SEQ ID NO:32)
  • Rv gDNA sequence 5'P-TAAGGGATTTTGGTCATGAGA 3' (SEQ ID NO:34)
  • FW ssDNA ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT (SEQ ID NO:35)
  • RV ssDNA ACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT (SEQ ID NO:36)
  • RV ssDNA AGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGT (SEQ ID NO: 40)
  • Rv gDNA sequence 5'P-TGTCTGTAAGCGGATGCCGGG 3' (SEQ ID NO:42)
  • RV ssDNA AGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTG (SEQ ID NO: 44)
  • aTF allosteric transcription factors
  • HosA derived from Escherichia coli UMN026 can specifically recognize p-hydroxybenzoic acid (p-HBA).
  • p-HBA is a phenolic acid that inhibits bacteria, fungi and enzymes. Preservatives in cosmetics.

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Abstract

一种新型中温原核Argonaute蛋白PbAgo表征及应用。具体地，一种用于检测靶标分子的检测体系，包括：(a)向导DNA(gDNA)；(b)核酸酶Argonaute(Ago)；和(c)报告核酸，其中若所述报告核酸被剪切，所述的剪切是可以被检测出的；其中，所述的靶标核酸分子为靶标DNA。并且还提供了相应的检测方法和检测试剂盒。该检测体系可快速、廉价并且高效地检测小分子。

Description

一种新型中温原核Argonaute蛋白PbAgo表征及应用 技术领域
本发明属于生物技术领域，具体涉及一种新型中温原核Argonaute蛋白PbAgo表征及应用。
背景技术
小分子检测在在环境监测、食品安全以及疾病诊断方面发挥着重要作用。快速、廉价和灵敏的检测技术的开发越来越受到人们的重视。
例如在食品领域中，常常会用到一些有助于防腐和抑菌的小分子；然而，若其含量超标，将会引起食物中毒或具有相应的食品安全隐患。为此，在食品安全的监管环节急需快速、廉价并且高效的小分子检测技术。
目前，小分子的常规检测主要依赖光谱和分离设备。然而，由于上述光谱或分离设备价格高昂，并且利用上述方法进行检测的操作较为繁琐；而对于环境监测、食品安全检测，以及疾病诊断的具体应用中，往往需要更加快捷的检测方法。因此，本领域已知的上述光谱分析等方法无法达到当前对于小分子检测的广大需求。
因此，本领域迫切需要开发一种快速、廉价并且高效的小分子检测技术。
发明内容
本发明的目的就是提供一种快速、廉价并且高效的小分子检测技术。
本发明具体地提供了一种基于中温原核Argonaute蛋白PbAgo的小分子检测方法。
在本发明的第一方面，提供了一种用于检测靶标分子的检测体系，所述检测体系包括：
(a)向导DNA(gDNA)；
(b)核酸酶Argonaute(Ago)；和
(c)报告核酸，其中若所述报告核酸被剪切，所述的剪切是可以被检测出的。
在另一优选例中，所述的靶标分子包括核酸分子和化学小分子。
在另一优选例中，所述的靶标分子是核酸分子，并且所述核酸分子可以和报告核 酸是同一种分子。
在另一优选例中，所述核酸分子是超螺旋状态的。
在另一优选例中，所述核酸分子中的GC含量≤36％，较佳地为≤20％，更佳地为≤14％。
在另一优选例中，当所述靶标分子是化学小分子时，所述的检测体系中还包括：(d)别构转录因子，所述的别构转录因子同时具有结合所述报告核酸和所述化学小分子的活性，并且所述报告核酸和所述化学小分子与所述别构转录因子的结合具有竞争性。
在另一优选例中，所述的别构转录因子选自下组：HosA、TetR、HucR等。
在另一优选例中，所述的化学小分子选自下组：对羟基苯甲酸(p-HBA)、四环素、尿酸等。
在另一优选例中，所述的别构转录因子是HosA，并且所述化学小分子是对羟基苯甲酸(p-HBA)。
在另一优选例中，所述的核酸酶Argonaute来源于北拟杆菌(Paenibacillus borealis)、丁酸梭菌(Clostridium butyricum)、产气荚膜梭菌(Clostridium perfringens)、玫瑰湖丝藻菌(Limnothrix rosea)、巴特勒肠杆菌(Intestinibacter bartlettii)、格氏嗜盐碱杆菌(Natronobacterium gregoryi)、嗜热古细菌(Pyrococcus furiosus)，或极端嗜热菌(Thermus thermophiles)。
在另一优选例中，所述的核酸酶Argonaute来源于北拟杆菌(Paenibacillus borealis)，所述的核酸酶Argonaute是核酸酶PbAgo。
在另一优选例中，所述的PbAgo包括野生型和突变型的PbAgo。
在另一优选例中，所述核酸酶PbAgo的包括如SEQ ID NO:1所示的氨基酸序列。
在另一优选例中，所述核酸酶的工作温度为10-65℃，较佳地为20-55℃，更佳地为30-45℃，更佳地为30-37℃。
在另一优选例中，所述的向导DNA是5’端磷酸化或羟基化的单链DNA分子。
在另一优选例中，所述的向导DNA是5’端磷酸化的单链DNA分子。
在另一优选例中，所述的向导DNA与所述的报告核酸之间具有互补的片段。
在另一优选例中，所述的向导DNA的长度为8-35nt，较佳地为14-21nt，最佳地为15-18nt。
在另一优选例中，所述向导DNA的5’端第一个核苷酸为磷酸化或羟基化修饰的胸腺嘧啶(T)。
在另一优选例中，所述向导DNA的核苷酸序列如SEQ ID NO:3所示。
在另一优选例中，所述的报告核酸是单链DNA(ssDNA)。
在另一优选例中，当所述报告核酸被剪切，所述的剪切能够通过电泳的方法被检测出。
在另一优选例中，所述的报告核酸是荧光报告核酸，所述荧光报告核酸带有荧光基团和/或淬灭基团。
在另一优选例中，所述的荧光基团和淬灭基团各自独立地位于所述荧光报告核酸的5’端、3’端。
在另一优选例中，所述的荧光基团和淬灭基团分别位于所述荧光报告核酸与所述向导DNA的互补区域的两侧。
在另一优选例中，所述的报告核酸是单链DNA(ssDNA)，长度为10-100nt，较佳地20-70nt，更佳地30-60nt，更佳地40-50nt，最佳地45nt。
在另一优选例中，所述荧光基团包括：FAM、HEX、CY5、CY3、VIC、JOE、TET、5-TAMRA、ROX、Texas Red-X，或其组合。
在另一优选例中，所述猝灭基团包括：BHQ、TAMRA、DABCYL、DDQ，或其组合。
在另一优选例中，所述的荧光基团是FAM。
在另一优选例中，所述的检测体系还包括：(e)二价金属离子。
在另一优选例中，所述的二价金属离子选自下组：Mg 2+、Mn 2+、Fe 2+、Co 2+、Cu 2+、Ni 2+，Zn 2+、Ca 2+，或其组合。
在另一优选例中，所述的二价金属离子选自下组：Mg 2+、Mn 2+，或其组合。
在另一优选例中，所述检测体系中，二价金属离子的浓度为10mM-3M，较佳地500mM-3M，更佳地1M-3M。
在另一优选例中，所述的检测体系还包括：(f)缓冲液。
在另一优选例中，所述缓冲液中，NaCl的浓度为≤750mM，较佳地为≤500mM，更佳地为≤100mM。
在另一优选例中，所述缓冲液的pH值为7-9，较佳地为8.0。
在另一优选例中，所述的检测体系还含有待检测的靶标分子。
在另一优选例中，所述的向导DNA与所述的报告核酸的序列互补结合后，引导所述PbAgo酶对所述报告核酸进行切割，从而产生可检测的信号(如荧光)。
在另一优选例中，所述的待检测的靶标分子在所述检测体系中的浓度为0.01-10μM，较佳地为0.05-1μM，更佳地为0.1μM。
在另一优选例中，所述的检测体系中，所述的报告核酸的浓度为0.01-10μM，较佳地为0.05-1μM，更佳地为0.1μM。
在另一优选例中，所述的检测体系中，所述核酸酶Ago的浓度为0.5-8μM，较佳地2-4μM，更佳地3μM。
在另一优选例中，所述的检测体系中，所述向导DNA的浓度为0.025-4μM，较佳地0.1-1μM，更佳地0.5μM。
在另一优选例中，所述的检测体系中，所述报告核酸与所述靶标分子的摩尔比为1:1.8至1:72000，较佳地1:36000。
在另一优选例中，所述的检测体系中，待检测的靶标分子、向导DNA、核酸酶Ago的摩尔比为1:(0.25-20):(1.5-120)，较佳地为1:(1-10):(5-50)，最佳地为1:5:30。
在本发明的第二方面，提供了一种用于检测靶标核酸分子的试剂盒，所述试剂盒包括：
(i)如本发明第一方面所述的检测体系或用于配制所述检测体系的试剂；和
(ii)使用说明书，所述说明书描述了用所述的检测体系检测靶标分子的方法。
在另一优选例中，所述的试剂盒包括：
(a)第一容器以及位于所述第一容器的向导DNA；
(b)第二容器以及位于第二容器的核酸酶Argonaute(Ago)；和
(c)第三容器以及位于第三容器的报告核酸。
在另一优选例中，所述的试剂盒还含有：
(d)第四容器以及位于第四容器的别构转录因子。
在另一优选例中，所述的试剂盒还含有：
(e)第五容器以及位于第五容器的二价金属离子。
在另一优选例中，所述的试剂盒还包括：
(f)第六容器以及位于第六容器的缓冲液。
在另一优选例中，所述第一容器、第二容器、第三容器、第四容器、第五容器和第六容器可以是相同或不同的容器。
在本发明的第三方面，提供了一种检测样本中是否存在靶标分子的方法，包括以下步骤：
(a)提供如本发明第一方面所述的用于检测靶标分子的检测体系；和
(b)将所述检测体系与待检测的样本在一定温度下进行反应，从而形成反应溶液；
(c)对所述反应溶液进行检测，从而获得剪切信号值；
其中，所述反应溶液中检测到剪切信号值，则表示所述样本中存在靶标分子；而所述反应溶液中没有检测到剪切信号值，则表示所述样本中不存在靶标分子。
在另一优选例中，所述的检测包括定性检测和定量检测。
在另一优选例中，所述步骤(c)中的检测可包括：利用电泳法对报告核酸进行长度的鉴定，从而判断所述的报告核酸是否被剪切。
在另一优选例中，在步骤(c)中所述的检测包括：采用酶标仪或者荧光分光光度计进行检测。
在另一优选例中，所述的方法是体外方法。
在另一优选例中，所述的方法是非诊断性和非治疗性的。
在本发明的第四方面，提供了一种核酸酶Argonaute的用途，用于制备检测靶标分子的试剂或试剂盒。
在另一优选例中，所述核酸酶Argonaute来源于北拟杆菌Paenibacillus borealis；或是其具备相同或相似功能的同源类似物。
在另一优选例中，所述的PbAgo包括野生型和突变型的PbAgo。
在另一优选例中，所述的核酸酶Argonaute具有选自下组的氨基酸序列：
(i)如SEQ ID NO:1所示的氨基酸序列；和
(ii)在如SEQ ID NO:1所示序列的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基，更佳地1至3个氨基酸残基)，从而获得的氨基酸序列；并且所述获得的氨基酸序列与如SEQ ID NO:1所示序列具有≥85％(优选地≥90％，更优选地≥95％，例如≥96％、≥97％、≥98％或≥99％)的序列同一性；并且所获得的氨基酸序列具备与(i)相同或相 似的功能。
本发明首先提供了一种编码中温原核Argonaute蛋白PbAgo的基因。通过与报道的CbAgo进行序列比对，选择序列一致性较高，并且来源于中温宿主的Ago蛋白，然后挖掘宿主北拟杆菌(Paenibacillus borealis strain)DSM 13188的全基因数据而获得，该基因全长2118bp，编码705个氨基酸。通过与已报道的原核Agos进行多重序列比对，PbAgo含有Ago蛋白发挥剪切活性的DEDX催化残基。
本发明构建了重组质粒pET-28a(+)-TEV-PbAgo，该重组质粒转化大肠杆菌BL21(DE3)，通过IPTG低温诱导表达，实现了PbAgo的异源活性表达，菌体破碎后的上清液通过Ni-NTA柱亲和纯化后，SDS-PAGE结果显示浓度较高的蛋白条带，分子量与目标蛋白分子量相符，表明PbAgo可以在大肠杆菌产生高效的可溶性表达。
本发明所得到的新型中温原核Argonaute蛋白PbAgo分子量约为80.79kDa，在5’P gDNA的指导下，最适反应温度在30℃-55℃之间；在5’OH gDNA的指导下，最适反应温度在55℃-65℃之间。二价金属离子如Mn 2+、Mg 2+都能够促进PbAgo发挥剪切活性，并且二价金属离子浓度在500mM至3M范围内，PbAgo酶活性保持高水平。该酶不耐受NaCl，NaCl会抑制PbAgo剪切ssDNA的活性。经过测定其剪切动力学看出，在5’P DNA guide的指导下，PbAgo反应很迅速，并且能够在30min内剪切95％的底物。其将在生物体内的基因编辑中具有广阔的前景。
本发明所得到的PbAgo能够在30℃结合gDNA，剪切不规则的双链DNA。别构转录因子HosA可结合不规则的dsDNA，小分子化合物p-HBA存在时，竞争结合HosA，释放出来的不规则dsDNA可被PbAgo剪切。可将PbAgo的这一特性结合HosA用于小分子化合物检测。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了PbAgo与已报道的原核Ago的多重序列比对结果。由图可知，PbAgo和已报道的其他原核Agos一样，也包含一个发挥剪切活性的DEDX活性中心。
图2显示了重组菌株表达PbAgo的SDS-PAGE电泳结果。其中，M是蛋白质标记(marker)；1是PbAgo纯化液。
图3显示了设计合成的45nt ssDNA/ssRNA靶标序列(target)和与之互补配对的21nt 5’P/OH修饰的DNA/RNA向导序列(guide)。
图4显示了纯化的PbAgo对向导序列(guide)和靶标序列(Target)的偏好性。由图可知，PbAgo可在37℃条件下，利用5’P/OH gDNA剪切与gDNA互补配对的ssDNA。
图5显示了在两种不同gDNA指导下PbAgo发挥剪切活性的最适温度。在5’P gDNA的指导下，最适反应温度在30℃-55℃之间；在5’OH gDNA的指导下，最适反应温度在55℃-65℃之间。
图6显示了在两种不同gDNA指导下PbAgo对二价金属离子的偏好性。由图可知，二价金属离子如Mn 2+、Mg 2+都能够促进PbAgo发挥剪切活性。
图7显示了在两种不同gDNA指导下，不同的二价金属离子(Mn 2+)浓度对PbAgo剪切活性的影响。由图可知，二价金属离子浓度在500mM至3M范围内，PbAgo酶活性保持高水平。
图8显示了在两种不同gDNA指导下PbAgo对NaCl的耐受性。由图可知，该酶不耐受NaCl，NaCl会抑制PbAgo剪切ssDNA的活性。
图9显示了在最适反应条件下测定的两种不同gDNA指导的PbAgo的剪切动力学。
图10显示了在最适反应条件下测定的两种不同gDNA指导的PbAgo在前20min的剪切动力学。由图可知，PbAgo在5’P gDNA指导下，剪切速率和剪切效率都更高。
图11显示了gDNA长度对PbAgo剪切活性的影响。由图可知，gDNA长度在13-30nt时，PbAgo具有剪切活性；gDNA长度在14-21nt时，PbAgo剪切效率较高；gDNA长度在15-18nt时，PbAgo剪切效率最好。
图12显示了PbAgo检测小分子化合物p-HBA的结果。其中4号样品在不加p-HBA的前提下，PbAgo/gDNA依然与HosA竞争结合dsDNA，与5号加p-HBA的样品相比，区别较小，造成检测的背景值过高。由此可知，HosA和PbAgo因共同的底物而相互干扰，后续可以针对这一问题做出相应调整。
图13显示了PbAgo在一对5’P gDNA的指导下，在37℃反应3h剪切质粒pUC19的结果。由图可知，7号样品，PbAgo可以利用一对5’P gDNA，分别靶向质粒的 一条链，剪切超螺旋状态的质粒，生成线性化质粒DNA。OC：开环质粒(质粒一条链断开)；LIN：线性化质粒(质粒双链断开)；SC：超螺旋质粒。
图14显示了PbAgo在一对5’OH gDNA的指导下，在65℃反应3h剪切质粒pUC19的结果。由图可知，7号样品，PbAgo可以利用一对5’OH gDNA，分别靶向质粒的一条链，剪切超螺旋状态的质粒，生成线性化质粒DNA。
图15显示了质粒目标片段GC含量对PbAgo剪切效率的影响。在质粒pUC19分别找到了6段50bp GC含量不同的目标片段，分别设计合成了6对与之互补配对的5’P gDNA。由图可知，50bp目标DNA片段的GC含量越低，PbAgo的剪切效果越好。PbAgo能够剪切GC含量不高于36％的双链DNA片段，生成线性化质粒；但尽管片段GC含量高达70％，PbAgo能使部分pUC19成为开环状态，可能是由于PbAgo在5’P gDNA指导下剪切了质粒的一条链。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种基于新型核酸酶Ago的靶标分子检测方法。具体地，本发明通过体外表达和纯化分离获得了核酸酶PbAgo，并且通过大量的摸索实验，获得了其最优的反应参数。实验结果表明，本发明核酸酶PbAgo的最适反应温度在30℃-65℃之间；二价金属离子如Mn 2+、Mg 2+，并且二价金属离子浓度在500mM至3M范围内能够显著促进PbAgo发挥剪切活性；本发明核酸酶不耐受NaCl。在5’P DNA引导序列的指导下，本发明PbAgo可在30℃下迅速反应，其能够在30min内剪切95％的底物(即不规则dsDNA)。此外，本发明的反应体系还可用于剪切超螺旋状态下的质粒DNA，有望为体内基因编辑提供新的酶资源。在此基础上完成了本发明。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属 领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。
如本文所用，术语“含有”或“包括(包含)”可以使开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”或“由…构成”。
“转导”、“转染”、“转化”或本文用到的术语指的是将外源多核苷酸传递导至宿主细胞，转录和翻译产生多肽产物的过程，包括利用质粒分子将外源多核苷酸引入宿主细胞(例如大肠杆菌)。
“基因表达”或“表达”指的是基因转录，翻译和翻译后修饰产生基因的RNA或蛋白产物的过程。
“多核苷酸”指的是任意长度的核苷酸的聚合形式，包括脱氧核苷酸(DNA)，核糖核苷酸(RNA)，其杂合序列和类似物。多核苷酸可包括修饰的核苷酸，比如甲基化或加帽的核苷酸或核苷酸类似物。本文使用的术语多核苷酸指可互换的单链和双链分子。除非另有说明，本文描述的任意实施例里的多核苷酸包括双链的形式和已知的或可预测的构成双链形式的两条互补的单链。
保守氨基酸的取代是本领域已知的。在一些实施例中，潜在的取代氨基酸在以下组的一个或多个内：甘氨酸，丙氨酸；和缬氨酸，异亮氨酸，亮氨酸和脯氨酸；天冬氨酸，谷氨酸；天冬酰胺，谷氨酰胺；丝氨酸，苏氨酸赖氨酸，精氨酸和组氨酸；和/或苯丙氨酸，色氨酸和酪氨酸；蛋氨酸和半胱氨酸。此外，本发明还提供了允许来自不同基团的氨基酸取代的非保守的氨基酸取代。
本领域技术人员将容易理解本文所述的所有参数，尺寸，材料和构造的含义。实际参数，尺寸，材料和/或配置取决于使用本发明说明的特定应用。本领域技术人员能够理解，实施例或权利要求仅是通过示例的方式给出的，并且在等效物或权利要求的范围内，本发明的实施例可涵盖的范围不限于具体描述和要求的范围。
本文的定义和使用的所有定义应被理解为超过词典定义或通过引用并入的文档中的定义。
本文所发明的所有参考文献，专利和专利申请都相对于其所引用的主题通过引用并入，在某些情况下可能包含整个文档。
应当理解，对于本文所述的包括一个以上步骤的任何方法，步骤的顺序不一定限于这些实施例中描述的顺序。
Ago酶
Argonaute蛋白属于PIWI(P element-induced wimpy testis)蛋白超家族，其由PIWI结构域的存在而界定，广泛存在于生活的所有领域，能够结合siDNA或siRNA指导链来特异性沉默或剪切互补核酸靶标链。研究表明，Ago在生物体细胞免疫防御及代谢调控中发挥重要作用，并可能具有人工基因编辑的应用潜力，因此针对Ago蛋白的功能研究成为生物学研究中新关注点。
Ago蛋白最初是在真核生物中发现的，是RNA干扰(RNAi)途径的关键参与者。真核Argonaute蛋白(eAgos)作为多蛋白RNA诱导沉默复合物(RISC)的核心，能够结合siRNA分子作为指导链，剪切互补的靶标RNA，直接沉默靶标RNA的翻译；或通过与靶标RNA结合，募集其他沉默因子来促进其降解，进而间接沉默靶标RNA。因此，eAgos可以在转录后调节基因表达，保护其宿主不受入侵RNA病毒的侵害，并通过降低转座子的流动性保持基因组的完整性。
Argonaute蛋白还存在于原核生物中。对一些原核Ago(pAgos)蛋白(主要来自嗜热细菌和古菌)的结构和生化研究表明，它们在体外可以发挥核酸内切酶作用，在体内可以发挥宿主防御作用。pAgos可以结合siDNA指导链来特异性剪切和指导链互补配对的DNA靶标链。截至2018年，已报道的pAgos主要来源于高温宿主，多用于基因检测。常温条件下活性很低，无法作为基因编辑的工具。2019年至今，陆续报道了一些来源于常温宿主的pAgos，能在常温条件下发挥DNA指导的DNA剪切活性，并且能够剪切GC含量较低的质粒。
如本文所用，术语“核酸酶Paenibacillus borealis”、“核酸酶Paenibacillus borealis”、“PbAgo酶”可互换使用，指本发明第一方面的检测体系中所述的核酸酶。
野生型的PbAgo酶具有如SEQ ID NO:1所示的氨基酸序列。
Figure PCTCN2022088007-appb-000001
Figure PCTCN2022088007-appb-000002
本发明的PbAgo酶还可包含其保留了功能活性的突变形式。所述的突变形式可含有在如SEQ ID NO:1所示序列的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基，更佳地1至3个氨基酸残基)，从而获得的氨基酸序列；并且所述获得的氨基酸序列与如SEQ ID NO:1所示序列具有≥85％(优选地≥90％，更优选地≥95％，例如≥96％、≥97％、≥98％或≥99％)的序列同一性；并且所获得的氨基酸序列具备与野生型PbAgo酶相同或相似的功能。
检测体系
如本文所用，术语“本发明检测体系”、“基于Argonaute蛋白的核酸检测体系”可互换使用，指本发明第一方面中所述的用于检测靶标分子的检测体系。在本发明的检测体系为基于本发明所提供的适用于本发明Ago酶(特别是PbAgo酶)的功能活性的反应条件而进行检测的。
在本发明中，提供了一种用于检测靶标分子的检测体系，所述检测体系包括：(a)向导DNA(gDNA)；(b)核酸酶Argonaute(Ago)；和(c)报告核酸，其中若所述报告核酸被剪切，所述的剪切是可以被检测出的；其中，所述的靶标核酸分子为靶标DNA。
在本发明的检测体系中，优选地，所述核酸酶的工作温度为10-65℃，较佳地为20-55℃，更佳地为30-45℃，更佳地为30-37℃。
优选地，所述的向导DNA是5’端磷酸化或羟基化的单链DNA分子；更加优选地，所述向导DNA是5’端磷酸化的单链DNA分子。优选地，所述的向导DNA的长度为8-35nt，较佳地为14-21nt，最佳地为15-18nt。
优选地，所述的报告核酸是单链DNA(ssDNA)。在一个实施方式中，当所述报告核酸被剪切，所述的剪切能够通过电泳的方法被检测出。
在另外的实施方式中，所述的报告核酸是荧光报告核酸，所述荧光报告核酸带有 荧光基团和淬灭基团。所述的荧光基团和淬灭基团各自独立地位于所述荧光报告核酸的5’端、3’端；优选地，所述的荧光基团和淬灭基团分别位于所述荧光报告核酸与所述向导DNA的互补区域的两侧。在另一优选例中，所述荧光基团包括：FAM、HEX、CY5、CY3、VIC、JOE、TET、5-TAMRA、ROX、Texas Red-X，或其组合。在另一优选例中，所述猝灭基团包括：BHQ、TAMRA、DABCYL、DDQ，或其组合。
所述的报告核酸的长度为10-100nt，较佳地20-70nt，更佳地30-60nt，更佳地40-50nt，最佳地45nt。
在另一个实施方式中，本发明检测体系中的报告核酸还可以和靶标分子是同一种分子。优选地，该核酸分子是超螺旋状态的质粒DNA。更加优选地，所述核酸分子中的GC含量不超过36％，较佳地不超过20％，更佳地不超过14％。
在本发明的检测体系还可包含二价金属离子。所述的二价金属离子选自下组：Mg 2+、Mn 2+、Fe 2+、Co 2+、Cu 2+、Ni 2+，Zn 2+、Ca 2+，或其组合。优选为：Mg 2+、Mn 2+，或其组合。在所述检测体系中，二价金属离子的浓度为10mM-3M，较佳地500mM-3M，更佳地1M-3M。
在一个实施方式中，本发明所述的检测体系中，NaCl的浓度为≤750mM，较佳地为≤500mM，更佳地为≤100mM。并且优选地，本发明所述检测体系的pH值为7-9，较佳地为8.0。
检测方法
如本文所用，术语“本发明检测方法”、“基于Argonaute蛋白的核酸检测方法”可互换使用，指本发明第三方面中所述的检测方法。
在本发明中，提供了一种检测样本中是否存在靶标分子的方法，包括以下步骤：(a)提供本发明所述的用于检测靶标分子的检测体系；和(b)将所述检测体系与待检测的样本在一定温度下进行反应，从而形成反应溶液；(c)对所述反应溶液进行检测，从而获得剪切信号值；其中，所述反应溶液中检测到剪切信号值，则表示所述样本中存在靶标分子；而所述反应溶液中没有检测到剪切信号值，则表示所述样本中不存在靶标分子。
所述的检测包括定性检测和定量检测。
在一个实施方式中，所述步骤(c)中的检测可包括：利用电泳法对报告核酸进行长度的鉴定，从而判断所述的报告核酸是否被剪切。若产生了较短长度的核酸产物， 这说明该报告核酸被检测体系中的Ago剪切。
在另一个实施方式中，若所述检测体系中报告核酸中带有荧光基团和/或淬灭基团，那么在步骤(c)中所述的检测包括：采用酶标仪或者荧光分光光度计进行检测。
在本发明的一个实施方式中，所述的方法是体外方法。在另一个实施方式中，所述的方法是非诊断性和非治疗性的。
试剂盒
在本发明中，提供了一种用于检测靶标核酸分子的试剂盒，包括：(i)本发明的检测体系或用于配制所述检测体系的试剂；和(ii)使用说明书，所述说明书描述了用所述的检测体系检测靶标分子的方法。
在具体的实施方式中，所述的试剂盒包括：(a)第一容器以及位于所述第一容器的向导DNA；(b)第二容器以及位于第二容器的核酸酶Argonaute(Ago)；和(c)第三容器以及位于第三容器的报告核酸。
优选地，所述的试剂盒还含有：(d)第四容器以及位于第四容器的别构转录因子。优选地，所述的试剂盒还含有：(e)第五容器以及位于第五容器的二价金属离子。
在本发明的各个实施方式中，上述各容器可以是相同或不同的容器。
本发明的主要优点包括：
1)本发明检测体系中的PbAgo可以用两种5’端不同修饰(5’P/OH)的gDNA剪切ssDNA。
2)本发明检测体系中的PbAgo在5’P gDNA指导下剪切ssDNA的反应速率很快,反应时间较短，只需30min就能达到近乎100％剪切。
3)本发明检测体系中的PbAgo对NaCl的耐受性较高，反应体系中NaCl浓度不高于750mM时，PbAgo都具有较高的剪切活性。
4)本发明检测体系中的PbAgo对gDNA长度的要求较低，gDNA为14-21nt时，PbAgo都具有较高的剪切活性。
5)本发明检测体系中的PbAgo能够在一对5’P/OH gDNA指导下剪切GC含量不高于36％的质粒dsNDA(pUC19)。
6)本发明首次将Ago和别构转录因子一起用来检测小分子化合物。PbAgo能够利用HosA的变构效应检测p-HBA，检测速率远高于本领域已知的其他检测方法。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：PbAgo基因的获得
在NCBI数据库(https://www.ncbi.nlm.nih.gov/)中通过BLAST对CbAgo的氨基酸序列进行相似性检索，选取部分序列一致性较高，并且来源于中温宿主的Agos。将氨基酸序列和已报道的原核Agos用MEGA X软件进行分析，构建同源进化树，选取与已报道的中温原核Agos亲缘关系近的PbAgo。然后通过与已报道的原核Agos进行多重序列比对分析，确定其是否包含DEDX催化残基。
部分序列的同源比对结果如图1所示。
实施例2：PbAgo异源表达
将PbAgo的基因序列构建到pET-28a(+)-TEV载体上，合成质粒。用合成的重组质粒转化大肠杆菌BL21(DE3)，然后从转化的平板上挑取单克隆转接试管进行培养，保存甘油菌。
重组菌株表达PbAgo的SDS-PAGE电泳结果如图2所示。
含有pET-28a(+)-TEV-PbAgo表达载体的菌液至接种于5mL LB液体培养基(含50μg/mL卡那霉素)中，220rpm、37℃过夜培养；以1％接种量转接至1L LB液体培养基(含50μg/mL卡那霉素)中，220rpm、37℃培养至OD 600达到0.6-0.8时，加入终浓度为0.5mM的IPTG，16℃、220rpm诱导表达16h-18h。
实施例3：PbAgo分离纯化
以6000rpm、4℃离心30min收集菌体，使用重悬缓冲液(含20mM Tris-HCl pH8.0、500mM NaCl、10mM咪唑、2％甘油)重悬菌体，然后用预冷的均质机高压破碎。破碎液通过10000rpm，4℃离心30min得到上清，再利用Ni-NTA柱纯化，得到PbAgo的纯化液，通过SDS-PAGE进行检测和鉴定。
纯化后的目的蛋白用超滤管于4℃、4500rpm浓缩至2.5mL，然后进一步纯化 除去咪唑。
酶浓度通过BCA试剂盒进行测定，测定步骤按照操作说明进行。以BSA作为标准品，配置标准溶液，绘制标准曲线，依此计算纯化的目的蛋白浓度，将目的蛋白保存于-80℃。
实施例4：PbAgo酶学性质研究
4.1 PbAgo的剪切活性测定
设计带有荧光修饰的ssDNA和ssRNA靶标核酸以及互补的四种gDNA和gRNA，并送金斯瑞生物科技有限公司合成。
设计合成的45nt ssDNA/ssRNA靶标序列(target)和与之互补配对的21nt5’P/OH修饰的DNA/RNA引导序列(guide)如图3所示。
45nt ssDNA序列(SEQ ID NO:2)：
Figure PCTCN2022088007-appb-000003
21nt DNA向导序列(SEQ ID NO:3)：
Figure PCTCN2022088007-appb-000004
45nt ssRNA序列(SEQ ID NO:4)：
Figure PCTCN2022088007-appb-000005
21nt RNA向导序列(SEQ ID NO:5)：
Figure PCTCN2022088007-appb-000006
在反应缓冲液(15mM Tris-HCl、pH8.0、200mM NaCl、2mM MnCl 2)中加入PbAgo(3μM)和ssDNA或ssRNA guide(0.5μM)，混匀，在常温下孵育15min。然后加入带荧光标记的ssDNA或ssRNA靶标链(target：guide：Ago的比例为1:5:30)，在37℃条件下反应30min。
反应结束后，取10μL样品，按1:1比例加入上样缓冲液(含95％(去离子)甲酰胺、0.5mM EDTA、0.025％溴酚蓝、0.025％二甲苯蓝)，在16％的核酸Urea-PAGE下进行电泳检测，并通过SYBR gold染色。
结果如图4所示。结果表明：PbAgo可在37℃条件下，利用5’P/OH gDNA剪切与gDNA互补配对的ssDNA。
4.2 PbAgo酶活力的影响
反应体系不变，在反应缓冲液(15mM Tris-HCl pH8.0、200mM NaCl、2mM MnCl 2) 中加入PbAgo和gDNA，混匀，在常温下孵育15min。然后加入荧光标记的ssDNA或ssRNA target，分别在10℃、20℃、30℃、37℃、45℃、55℃、65℃、75℃、85℃、95℃条件下反应30min。
反应产物在16％的核酸Urea-PAGE下进行电泳检测。凝胶通过SYBR gold染色后，用凝胶定量软件ImageJ进行胶图定量分析，并通过GraphPad Prism 8作图。
结果如图5所示。结果表明，在5’P gDNA的指导下，最适反应温度在30℃-55℃之间；在5’OH gDNA的指导下，最适反应温度在55℃-65℃之间。
4.3二价金属离子对PbAgo酶活力的影响
在反应缓冲液(15mM Tris-HCl pH8.0、200mM NaCl)中，分别加入终浓度为2mM的MgCl 2、MnCl 2、FeCl 2、CoCl 2、CuCl 2、NiCl 2，ZnCl 2、CaCl 2。然后加入PbAgo和gDNA，混匀，在常温下孵育15min。然后加入荧光标记的ssDNA或ssRNA target，在37℃条件下反应30min。将不加入金属离子的样品作为对照组。
反应产物如按上述方法进行检测、染色、胶图定量和作图。
结果如图6所示。结果表明：二价金属离子如Mn 2+、Mg 2+都能够促进PbAgo发挥剪切活性。
反应条件不变，在反应体系中分别加入不同浓度的MnCl 2：0mM、5mM、10mM、25mM、50mM、100mM、250mM、500mM、1000mM、2000mM、3000mM，检测在不同guides指导下PbAgo的最适MnCl 2浓度。
按上述方法进行胶图染色和定量作图。
结果如图7所示。结果表明，二价金属离子浓度在500mM至3M范围内，PbAgo酶活性保持高水平。
4.4 NaCl对PbAgo酶活力的影响
反应条件不变，在反应体系中分别加入不同浓度的NaCl：50mM、100mM、250mM、500mM、750mM、1000mM、1500mM、2000mM、2500mM、3000mM。检测在不同guides指导下，NaCl对PbAgo酶活力的影响。按上述方法进行胶图染色和定量作图。
结果如图8所示。结果表明， PbAgo酶不耐受NaCl，NaCl会抑制PbAgo剪切ssDNA的活性。
4.5两种gDNA指导下PbAgo的剪切动力学
PbAgo、gDNA和ssDNA浓度不变，在50mM NaCl和2mM MnCl 2浓度下，37℃反应不同时间：0min、3min、5min、10min、20min、30min、45min、60min、80min、100min、120min、150min和180min。在16％的核酸Urea-PAGE下进行电泳检测。按上述方法进行胶图染色和定量作图。
两种不同gDNA指导的PbAgo的剪切动力学结果如图9和10所示。结果表明，PbAgo在5’P gDNA指导下，剪切速率和剪切效率都更高。
4.6 gNDA长度对PbAgo酶活力的影响
根据带有荧光修饰的ssDNA靶标核酸，设计与之配对不同长度的5’P gDNA。
不同长度的5’P gDNA序列：
35nt gDNA：TGAGGTAGTAGGTTGTATAGTATATTAAATTATTT(SEQ ID NO:6)
30nt gDNA：TGAGGTAGTAGGTTGTATAGTATATTAAAT(SEQ ID NO:7)
25nt gDNA：TGAGGTAGTAGGTTGTATAGTATAT(SEQ ID NO:8)
21nt gDNA：TGAGGTAGTAGGTTGTATAGT(SEQ ID NO:9)
18nt gDNA：TGAGGTAGTAGGTTGTAT(SEQ ID NO:10)
16nt gDNA：TGAGGTAGTAGGTTGT(SEQ ID NO:11)
15nt gDNA：TGAGGTAGTAGGTTG(SEQ ID NO:12)
14nt gDNA：TGAGGTAGTAGGTT(SEQ ID NO:13)
13nt gDNA：TGAGGTAGTAGGT(SEQ ID NO:14)
12nt gDNA：TGAGGTAGTAGG(SEQ ID NO:15)
10nt gDNA：TGAGGTAGTA(SEQ ID NO:16)
8nt gDNA：TGAGGTAG(SEQ ID NO:17)
反应体系中的其他成分和反应条件不变，在体系中加入0.5μM不同长度的5’P gDNA，在37℃条件下反应30min。将不加入gDNA的样品作为对照组。反应产物如按上述方法进行检测、染色、胶图定量和作图。
结果如图11所示，gDNA在长度14-21nt范围内时PbAgo的剪切效率保持较高水平，更佳地为15-18nt；gDNA长度为16nt时，PbAgo的剪切效率最高。而后随着gDNA的长度增加，PbAgo的剪切效率降低。
4.7 PbAgo检测p-HBA
先在反应体系中加入不规则dsDNA和别构转录因子HosA，30℃孵育20min，使之结合。再将目标小分子p-HBA添加至反应体系中，诱导dsDNA与HosA解离，进而通过PbAgo对游离的dsDNA进行检测。
PbAgo检测小分子化合物p-HBA的结果如图12所示。
从结果可看出，当体系中不存在小分子p-HBA时(对应于泳道4和泳道6)，dsDNA能够部分被PbAgo剪切，而这种情况下的剪切产物信号为背景信号；而当体系中存在小分子p-HBA时(对应于泳道5)，PbAgo对dsDNA的剪切产物信号大大提高，显著高于背景信号。因此，本发明基于PbAgo酶的方法可以用来检测样品中的小分子化合物p-HBA。
4.8 PbAgo剪切质粒dsDNA
设计合成与pUC19片段互补配对的一对5’P/OH gDNA，送生工生物工程科技有限公司合成。
pUC19序列：
Figure PCTCN2022088007-appb-000007
Figure PCTCN2022088007-appb-000008
pUC19目标片段序列(50bp)：
FW ssDNA：TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAA(SEQ ID NO:19)
RV ssDNA：TTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA(SEQ ID NO:20)
21nt FW gDNA序列：
5’P/OH-TTAGATTGATTTAAAACTTCA 3’(SEQ ID NO:21)
21nt RV gDNA序列：
5’P/OH-TGAAGTTTTAAATCAATCTAA 3’(SEQ ID NO:22)
在反应缓冲液(15mM Tris-HCl、pH 8.0、2mM MnCl 2)中加入3μM  PbAgo和一对0.5μM 5’P/OH gDNA，混匀，在常温下孵育15min。然后加入600ng pUC19质粒，在37℃/65℃条件下反应3h。反应结束后，取10μL样品，按5:1比例加入上样缓冲液，用1％的琼脂糖凝胶进行电泳检测。
PbAgo在5’P/OH gDNA指导下剪切pUC19的结果如图13、14所示。
结果表明，在体系中只加入PbAgo，可能通过切开一条链，将质粒底物从超螺旋状态转变为开环状态，但未观察到质粒DNA的显著线性化或降解。当质粒被结合了单个gDNA的PbAgo靶向时，可还观察到超螺旋的减少。当使用两种PbAgo-gDNA复合物时，每种都靶向质粒的一条链，可以观察到一部分线性化的目标质粒DNA。
这暗示着PbAgo-gDNA复合物介导的每条靶标质粒DNA链的切口导致了双链DNA断裂的产生，并且5'P gDNA引导PbAgo剪切pUC19的效率比5'OH gDNA高。
4.9质粒目标片段GC含量对PbAgo剪切质粒的影响
PbAgo和质粒浓度不变，加入和不同GC含量片段互补配对的一对gDNA，在50mM NaCl和2mM Mn 2+浓度下，在37℃反应3h，测定目标片段GC含量对Ago剪切质粒DNA的活性的影响。反应结束后，取10μL样品，按5:1比例加入上样缓冲液，用1％的琼脂糖凝胶进行电泳检测。
不同GC含量目标片段(50bp)：
14％GC：
FW ssDNA：TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAA(SEQ ID NO:23)
RV ssDNA：TTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA(SEQ ID NO:24)
FW gDNA序列：5’P-TTAGATTGATTTAAAACTTCA 3’(SEQ ID NO:25)
Rv gDNA序列：5’P-TGAAGTTTTAAATCAATCTAA 3’(SEQ ID NO:26)
20％GC：
FW ssDNA:ATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGT(SEQ ID NO:27)
RV ssDNA:ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT(SEQ  ID NO:28)
FW gDNA序列：5’P-TTAAAACTTCATTTTTAATTT 3’(SEQ ID NO:29)
Rv gDNA序列：5’P-AAATTAAAAATGAAGTTTTAA 3’(SEQ ID NO:30)
36％GC：
FW ssDNA:GATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGT(SEQ ID NO:31)
RV ssDNA:ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC(SEQ ID NO:32)
FW gDNA序列：5’P-TCTCATGACCAAAATCCCTTA 3’(SEQ ID NO:33)
Rv gDNA序列：5’P-TAAGGGATTTTGGTCATGAGA 3’(SEQ ID NO:34)
50％GC：
FW ssDNA:ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT(SEQ ID NO:35)
RV ssDNA:ACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT(SEQ ID NO:36)
FW gDNA序列：5’P-CGTGAGTTTTCGTTCCACTGA 3’(SEQ ID NO:37)
Rv gDNA序列：5’P-TCAGTGGAACGAAAACTCACG 3’(SEQ ID NO:38)
60％GC：
FW ssDNA:ACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCT(SEQ ID NO:39)
RV ssDNA:AGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGT(SEQ ID NO:40)
FW gDNA序列：5’P-CCCGGCATCCGCTTACAGACA 3’(SEQ ID NO:41)
Rv gDNA序列：5’P-TGTCTGTAAGCGGATGCCGGG 3’(SEQ ID NO:42)
70％GC：
FW ssDNA:CAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCT(SEQ ID NO:43)
RV ssDNA:AGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTG(SEQ ID NO:44)
FW gDNA序列：5’P-CGCCCTGACGGGCTTGTCTGC 3’(SEQ ID NO:45)
Rv gDNA序列：5’P-GCAGACAAGCCCGTCAGGGCG 3’(SEQ ID NO:46)
结果如图15所示，50bp目标DNA片段的GC含量越低，PbAgo的剪切效果越好。PbAgo能够剪切GC含量不高于36％的双链DNA片段，但尽管片段GC含量高达70％，PbAgo能使部分pUC19形成开环。
讨论
2019年，有文献报道利用别构转录因子(allosteric transcription factor，aTF)的别构效应可实现对目标小分子简单、灵敏、快速和低成本的检验。aTF可将难以检测的小分子信号转换成极易检测的DNA信号，是一种极具开发价值的小分子检测生物识别元件。来源于大肠杆菌UMN026的别构转录因子HosA能够特异性识别对羟基苯甲酸(p-HBA)，p-HBA是酚酸类物质，具有抑制细菌、真菌和酶的作用，常用作食品、药品、化妆品中的防腐剂。
而在本发明中，通过对PbAgo酶的功能活性的摸索和反应条件的优化，利用HosA的别构效应和PbAgo结合siDNA指导链剪切DNA靶标链的特点，开发和提供了了一种更为简单、更为快捷、低成本的p-HBA检测平台。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种用于检测靶标分子的检测体系，其特征在于，所述检测体系包括：
    (a)向导DNA(gDNA)；
    (b)核酸酶Argonaute(Ago)；和
    (c)报告核酸，其中若所述报告核酸被剪切，所述的剪切是可以被检测出的。
  2. 如权利要求1所述的检测体系，其特征在于，所述的靶标分子是核酸分子，并且所述核酸分子可以和报告核酸是同一种分子；
    或者，所述靶标分子是化学小分子，并且所述的检测体系中还包括：(d)别构转录因子，所述的别构转录因子同时具有结合所述报告核酸和所述化学小分子的活性，并且所述报告核酸和所述化学小分子与所述别构转录因子的结合具有竞争性。
  3. 如权利要求2所述的检测体系，其特征在于，所述的别构转录因子选自下组：HosA、TetR、HucR等。
  4. 如权利要求2所述的检测体系，其特征在于，所述的化学小分子选自下组：对羟基苯甲酸(p-HBA)、四环素、尿酸等。
  5. 如权利要求1所述的检测体系，其特征在于，所述的核酸酶Argonaute来源于北拟杆菌(Paenibacillus borealis)，所述的核酸酶Argonaute是核酸酶PbAgo。
  6. 如权利要求1所述的检测体系，其特征在于，所述核酸酶PbAgo包括如SEQ ID NO:1所示的氨基酸序列。
  7. 如权利要求1所述的检测体系，其特征在于，所述核酸酶的工作温度为10-65℃，较佳地为20-55℃，更佳地为30-45℃，更佳地为30-37℃。
  8. 如权利要求1所述的检测体系，其特征在于，所述的向导DNA是5’端磷酸化或羟基化的单链DNA分子。
  9. 如权利要求1所述的检测体系，其特征在于，所述的向导DNA与所述的报告核酸之间具有互补的片段。
  10. 如权利要求1所述的检测体系，其特征在于，所述的向导DNA的长度为8-35nt，较佳地为14-21nt，最佳地为15-18nt。
  11. 如权利要求1所述的检测体系，其特征在于，所述的检测体系还包括：(e)二价金属离子。
  12. 一种用于检测靶标核酸分子的试剂盒，其特征在于，所述试剂盒包括：
    (i)如权利要求1所述的检测体系或用于配制所述检测体系的试剂；和
    (ii)使用说明书，所述说明书描述了用所述的检测体系检测靶标分子的方法。
  13. 一种检测样本中是否存在靶标分子的方法，其特征在于，包括以下步骤：
    (a)提供如权利要求1所述的用于检测靶标分子的检测体系；和
    (b)将所述检测体系与待检测的样本在一定温度下进行反应，从而形成反应溶液；
    (c)对所述反应溶液进行检测，从而获得剪切信号值；
    其中，所述反应溶液中检测到剪切信号值，则表示所述样本中存在靶标分子；而所述反应溶液中没有检测到剪切信号值，则表示所述样本中不存在靶标分子。
  14. 一种核酸酶Argonaute的用途，其特征在于，用于制备检测靶标分子的试剂或试剂盒。
  15. 如权利要求14所述的用途，其特征在于，所述的核酸酶Argonaute具有选自下组的氨基酸序列：
    (i)如SEQ ID NO:1所示的氨基酸序列；和
    (ii)在如SEQ ID NO:1所示序列的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至10个氨基酸残基(较佳地1至5个氨基酸残基，更佳地1至3个氨基酸残基)，从而获得的氨基酸序列；并且所述获得的氨基酸序列与如SEQ ID NO:1所示序列具有≥85％(优选地≥90％，更优选地≥95％，例如≥96％、≥97％、≥98％或≥99％)的序列同一性；并且所获得的氨基酸序列具备与(i)相同或相似的功能。
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