Enzyme cascade amplification regulated and controlled Thermus thermophilus Argonaute biosensor, and preparation method and application thereof

Abstract

The invention discloses an enzyme cascade amplification regulated and controlled Thermus thermophilus Argonaute biosensor as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, incubating a reaction solution containing piRNA-36026 / piRNA-36743, a 3WJ template, a 3WJ primer, a linear template, an NEBuffer 2 buffer solution, an rCutSmart buffer solution, a dNTPs mixed solution, a KF polymerase and an Nt.BbvCI nicking endonuclease; and step 2, adding a reaction product in the step 1 into a reaction solution containing a signal probe, a # imgabs0 # reaction buffer solution and Tth Argonaute protein, and incubating. According to the invention, the high efficiency of enzyme cascade amplification and the accurate recognition and multi-round cutting activity of TtAgo are fully utilized, and ultrahigh sensitivity, good selectivity and strong multi-analysis capability are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of biological analysis, and particularly relates to an enzyme cascade amplification-regulated Thermus thermophilus Argonaute biosensor for simultaneously detecting a variety of PIWI-interacting RNAs. Background Art

[0002] Piwi-interacting RNAs (piRNAs) are small noncoding RNAs consisting of 23-31 nucleotides. Since their first discovery in 2006, they have been mainly involved in the regulation of gene expression and the maintenance of genome integrity. Unlike microRNAs (miRNAs) and small interfering RNAs (siRNAs), piRNAs are Dicer-independent and are produced from single-stranded precursor transcripts. piRNAs can specifically bind to Argonaute / PIWI family proteins PIWI proteins to form PIWI-piRNA complexes, thereby affecting piRNA biogenesis, transposon silencing, spermatogenesis, chromatin rearrangement, transcriptional and epigenetic regulation, and cell maintenance. Many studies have shown that piRNAs play key roles in various physiological processes, including germ cell development, neuronal function, and immune response. They are also associated with pathological conditions such as cancer, reproductive diseases, retinal diseases, cardiovascular diseases, and neurodegenerative diseases. In addition, abnormal expression of piRNAs has been observed in many cancers, including breast cancer, colorectal cancer, lung cancer, gastric cancer, glioma, and lymphoma. Notably, 2′-O-methylation at the 3′ end of piRNAs can resist oxidation and degradation, making them stable in human body fluids such as urine, serum, and plasma samples. Therefore, piRNAs can serve as non-invasive biomarkers and therapeutic targets, and accurate detection of piRNAs is crucial to advance our understanding of piRNA-related biomedical research and clinical diagnosis.

[0003] Traditional methods for detecting piRNAs include Northern blot, microarray, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). However, these methods have some inherent disadvantages, such as hazardous radioactive labels, cumbersome experimental operations, complex data analysis, large sample consumption, and poor reproducibility and sensitivity. Alternatively, several nucleic acid-based amplification strategies, including rolling circle amplification (RCA), ligase chain reaction (LCR), double-stranded specific nuclease (DSN)-assisted signal amplification, and catalytic hairpin assembly (CHA), have been introduced for the detection of piRNAs. Despite the advantages of simple operation and improved sensitivity, these strategies are limited to the detection of a single type of piRNA. Considering that a disease is often associated with different types of piRNAs, the combination of multiple piRNAs can greatly improve diagnostic accuracy. Therefore, the development of new methods for the simultaneous detection of multiple piRNAs remains a huge challenge.

[0004] Recently, endonucleases with sequence-specific catalytic properties have emerged as valuable tools for the identification of target nucleic acids. In particular, Argonaute (Ago) proteins, named for their homology to the Ago family of proteins in eukaryotes, are involved in RNA interference (RNAi) and small RNA-guided gene regulation. These proteins are found in a variety of organisms (e.g., eukaryotes, prokaryotes, and archaea), highlighting their evolutionary conservation and functional importance. The strict DNA-guided target recognition and cleavage activity of Ago proteins have enabled their application in the field of bioanalysis. An attractive feature of Ago proteins is the ability to cleave target nucleic acids without the need for a specific sequence motif, unlike the protospacer adjacent motif (PAM) involved in Cas proteins. Ago proteins can specifically cleave target nucleic acids by base pairing with a stretch of guide DNA (gDNA) between positions 10 and 11, providing the adaptability of less restrictive nucleic acid selection. Thermophilic variants of these proteins, derived from thermophilic microorganisms, add another layer of advantage based on their tolerance to high temperatures and enhance the robustness of Ago-based diagnostics. In addition, the use of single-stranded DNA (ssDNA) primers is more stable and less expensive than ssRNA primers, which can further improve detection efficiency. Summary of the invention

[0005] The purpose of the present invention is to provide an enzyme cascade amplification-regulated Thermus thermophilus Argonaute biosensor and its preparation method and use, by constructing an enzyme cascade amplification-regulated TtAgo biosensor for simultaneously detecting piR-36026 and piR-36743 in clinical breast tissue.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing a Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification comprises the following steps:

[0008] Step 1, incubate a reaction solution containing piRNA-36026 / piRNA-36743, 3WJ template, 3WJ primer, linear template, NEBuffer2 buffer, rCutSmart buffer, dNTPs mixed solution, KF polymerase, and Nt.BbvCI nicking endonuclease;

[0009] Among them, piRNA is a ribonucleic acid that interacts with PIWI protein; the " / " in piRNA-36026 / piRNA-36743 represents one of or, and;

[0010] 3WJ means three-way connection;

[0011] KF polymerase is an N-terminal truncation of DNA polymerase I that retains DNA polymerase activity but loses 5′→3′ exonuclease activity;

[0012] Nt.BbvCI nicking endonuclease can specifically cut one of the strands on the double-stranded DNA substrate, and the resulting DNA molecule is "nicked" rather than cut;

[0013] NEBuffer 2 buffer is the reaction buffer that comes with the KF polymerase product;

[0014] rCutSmart buffer is the reaction buffer that comes with the Nt.BbvCI nicking endonuclease product;

[0015] The dNTP mixture is an equimolar solution of ultrapure dATP, dCTP, dGTP, and dTTP;

[0016] Step 2, adding the reaction product of step 1 to a Incubate in a reaction buffer and a reaction solution of TthArgonaute protein to obtain a Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification;

[0017] Among them, Tth Argonaute (TtAgo) is a DNA-guided nuclease that cuts the complementary sequence of the guide oligonucleotide. The cleavage site corresponds to a signal probe that is a DNA sequence modified with Cy5 or Cy3 at one end and a quencher group at the other end.

[0018] The reaction buffer is the reaction buffer that comes with the TthArgonaute protein product.

[0019] In the step 1, 10 μL of a reaction solution containing different concentrations of piRNA-36026 / piRNA-36743, 100 nM 3WJ template, 100 nM 3WJ primer, 100 nM linear template, 1×NEBuffer 2, 1×rCutSmart buffer, 200 μM dNTPs mixed solution, 2 UKF polymerases, and 8 UNt.BbvCI nicking endonucleases was incubated at 37° C. for 60 minutes.

[0020] In step 2, the reaction product was added to a solution containing 200 μM signal probe, 1× The cells were incubated at 75°C for 30 minutes in a reaction buffer and 20 μL of a reaction solution containing 40 nM Tth Argonaute protein.

[0021] The NEBuffer 2 buffer solution includes 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM DTT.

[0022] The rCutSmart buffer includes 500 mM potassium acetate, 200 mM triacetin, 100 mM magnesium acetate, 1 mg / ml recombinant albumin, and has a pH of 7.9.

[0023] Said The reaction buffer included Tris-HCl, (NH4)2SO4, KCl, MgSO4, and 1% polyoxyethylene glycol p-isooctylphenyl ether.

[0024] The sequence of piRNA-36026 is: GGC CCCAUG GUG UAAUGG UCAGCACUC;

[0025] The sequence of piRNA-36743 is: GUU UCC GUA GUG UAG UGG UCA UCA CGUUCG CC;

[0026] The 3WJ templates include 3WJ template-36026 and 3WJ template-36743.

[0027] The sequence of the 3WJ template-36026 is: ACA GGC CAG GAG CTGAGGAAC CAAGAGGCT GAG GATCTTATC CTT TTACAC CAT GGG G;

[0028] The sequence of the 3WJ template-36743 is: CAA CAA GAA CTG CTG AGG AAA GTC TGTGCT GAGGAT CTTATC CTT CAC TACACTACG GA.

[0029] The 3WJ primers include 3WJ primer-36026 and 3WJ primer-36743,

[0030] The sequence of the 3WJ primer-36026 is: GAG TGC TGACCAGGATAA;

[0031] The sequence of the 3WJ primer-36743 is: GCGAAC GTGATG CAG GATAA.

[0032] The linear templates include linear template-36026 and linear template-36743,

[0033] The linear template-36026 sequence is: ACA GGC CAG GAG CTG AGG AAC CAA GAG GCT GA;

[0034] The linear template -36743 sequence is: CAA CAA GAA CTG CTG AGG AAA GTC TGT GCT GA.

[0035] The signal probes include signal probe-36026 and signal probe-36743,

[0036] The signal probe-36026 sequence is: Cy3-ACA GGC CAG GAG CTG A-BHQ2;

[0037] The sequence of the signal probe-36743 is: Cy5-CAA CAA GAA CTG CTG A-BHQ3.

[0038] The invention discloses a Thermus thermophilus Argonaute biosensor controlled by enzyme cascade amplification obtained by the preparation method.

[0039] The enzyme cascade amplification-regulated Thermus thermophilus Argonaute biosensor is used as a biosensor for simultaneously detecting multiple piRNAs. The detection limit of the biosensor for piR-36026 is 8.51aM, and the detection limit of piR-36743 is 13.34aM.

[0040] Beneficial effects: The present invention provides a Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification, and a preparation method and use thereof. This TtAgo biosensor has significant advantages such as simple design, high amplification efficiency and throughput, and good specificity. This TtAgo biosensor can greatly increase the amount of gDNA through enzyme cascade amplification, which helps to release abundant Cy3 / Cy5 molecules to produce enhanced fluorescence signals. Compared with the classic EXPER using SYBR dyes that can only detect a single target RNA, this TtAgo biosensor is able to detect multiple piRNAs simultaneously. CRISPR / Cas nucleases involve long and easily degradable RNA guides, while TtAgo only requires 16nt DNA guides, thereby reducing costs and improving detection stability. This TtAgo biosensor can easily detect any RNA target of interest because it is not restricted by sequences such as PAM motifs, increasing its versatility in diagnostic applications. By cleverly combining the unique features of TtAgo, such as efficient enzyme cascade amplification, programmability, and multi-flip cleavage activity, as well as the high signal-to-noise ratio of single-molecule detection, the TtAgo biosensor exhibits excellent specificity and high sensitivity over a large dynamic range of 6 orders of magnitude, with a detection limit of 8.51aM for piR-36026 and 13.34aM for piR-36743. In addition, the TtAgo biosensor can distinguish cancer cells and normal cells with high expression of piRNAs, and simultaneously quantify multiple piRNAs in cancer cells at the single-cell level, and can be further used to accurately distinguish cancer patients from healthy donors. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Biosensor principle for simultaneous detection of multiple Piwi-interacting RNAs.

[0042] Figure 2: A is a 14% non-denaturing PAGE analysis of the enzyme cascade amplification products induced by piR-36026, M lane: 20bp DNA ladder marker; Lane 1, 3WJ template-36026+3WJ primer-36026+linear template-36026+KF polymerase+Nt.BbvCI; Lane 2, piR-36026+3WJ template-36026+3WJ primer-36026+linear template-36026+KF polymerase+Nt.BbvCI; Lane 3, synthetic gDNA 1; B is a 14% non-denaturing PAGE analysis of the enzyme cascade amplification products induced by piR-36743, M lane: 20bp DNA ladder marker; Lane 1, 3WJ template-36743+3WJ primer-36743+linear template-36743+KF polymerase+Nt.BbvCI; Lane 2, piR-36743+3WJ template-36743+3WJ primer 36743+linear template-36743+FF polymerase+Nt.BbvCI; Lane 3, synthetic gDNA2; C is the Cy3 fluorescence emission spectrum in the presence (green line) and absence (gray line) of piR-36026, and the inset shows the Cy3 fluorescence image of the amplified product; D is the Cy5 fluorescence emission spectrum in the presence (red line) and absence (blue line) of piR-36743, and the inset shows the Cy5 fluorescence image of the amplified product.

[0043] Figure 3 : A to H are TIRF-based single-molecule fluorescence imaging in the presence (B and F), piR-36743 (C and G), and piR-36026+piR-36744 (D and H) and absence (A and E), respectively; I is the single-molecule stepwise photobleaching trajectory of a single Cy3 molecule over time; J is the single-molecule stepwise photobleaching trajectory of a single Cy5 molecule over time, the Cy3 fluorescence signal is shown in green, and the Cy5 fluorescence signal is shown in red, scale bar: 5 μm.

[0044] Figure 4 : A shows the changes in Cy3 counts induced by different concentrations of piR-36026; B shows the changes in Cy5 counts induced by different concentrations of piR-36743; C shows that Cy3 counts are linearly correlated with the logarithm of piR-36026 concentration; D shows that Cy5 counts are linearly correlated with the logarithm of piR-36743 concentration. Data are expressed as mean ± standard deviation (n = 3).

[0045] Figure 5: Cy3 (green column) and Cy5 (red column) counts induced by piR-36026+piR-36743, piR-36026, piR-36744, circMTO1, piR-651, piR-932, DSCAM-AS1, miR-10b, miR-210 and control groups were measured respectively; Inset: The corresponding radar graphs of Cy3 and Cy5 counts generated by the same batch of samples mentioned above. Data are expressed as mean ± standard deviation (n = 3).

[0046] Figure 6 :A is a schematic diagram of cell piRNAs analysis; B and C are the measurement of piR-36026 (B) and piR-36743 (C) in different cell lines using TtAgo biosensor and qRT-PCR. D is the linear relationship between Cy3 counts and the logarithm of MCF-7 cell number from 1 to 100,000 cells; E is the linear relationship between Cy5 counts and the logarithm of BT474 cell number from 1 to 100,000 cells, and the data are expressed as mean ± standard deviation (n = 3).

[0047] Figure 7 : A and B are heat maps (A) and box plots (B) of piR-36026 analysis in breast cancer patient tissues and healthy donor tissues by the TtAgo biosensor and qRT-PCR; C and D are heat maps (C) and box plots (D) of piR-36743 analysis in breast cancer patient tissues and healthy donor tissues by the TtAgo biosensor and qRT-PCR. DETAILED DESCRIPTION

[0048] The present invention will be further explained below in conjunction with the accompanying drawings and embodiments.

[0049] Reagents and Materials:

[0050] All HPLC-purified oligonucleotides were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). TthArgonaute (TtAgo), 10× Reaction buffer (200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 1% X-100, pH 8.8), Nt.BbvCI cutting endonuclease, 10×rCutSmart TMBuffer (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1 mg / ml recombinant albumin, pH 7.9 dNTP) solution mixture was purchased from New England Biolabs (Ipswich, MA, USA). SYBR Gold was purchased from Invitrogen Co. (Carlsbad, CA, USA). Diethylpyrocarbonate (DEPC) treated water (RNase-free) was purchased from Sanguang Biotechnology Co., Ltd. Human breast adenocarcinoma cell lines (MCF-7 cells and BT-474 cells), human breast normal cell line (MCF-10A cells), human lung adenocarcinoma cell line (A549 cells), and human cervical cancer cell line. Formalin-fixed paraffin-embedded (FFPE) breast tissue sections were obtained from Nanjing Drum Tower Hospital (Nanjing, Jiangsu, China), and the experiments were approved by the Ethics Committee of Nanjing Drum Tower Hospital.

[0051] Fluorescence spectrum detection and actual sample measurement:

[0052] Fluorescence emission spectra were obtained at room temperature using FLS1000 (Edinburgh Instruments, UK) with a xenon lamp as the excitation source. The emission and excitation slits were both 5 nm, and the excitation wavelength was 488 nm. Data analysis was performed using the Cy3 fluorescence intensity at 565 nm and the Cy5 fluorescence intensity at 667 nm, respectively. The SanPrep column microRNA extraction kit (Sangon Biotech Co., Ltd., Shanghai) was used to obtain cell lysates from human tissues. The resulting extracts were immediately used to detect piRNA activity.

[0053] Gel electrophoresis:

[0054] The enzyme cascade amplification products were analyzed by 14% non-denaturing polyacrylamide gel (PAGE) at a constant voltage of 110 V for 65 min in 1× TBE buffer (9 mM Tris-HCl, 9 mM boric acid, 0.2 mM EDTA, pH 7.9) with 1× SYBR Gold as a fluorescent indicator. Subsequently, the gel was imaged by the Bio-Rad ChemiDoc MP imaging system (Hercules, California, USA).

[0055] Embodiment 1:

[0056] Step 1, incubate 10 μL of reaction solution containing different concentrations of piRNA-36026 / piRNA-36743, 100 nM 3WJ template, 100 nM 3WJ primer, 100 nM linear template, 1×NEBuffer 2, 1×rCutSmart buffer, 200 μM dNTPs mixture, 2 U KF polymerase, and 8 U Nt.BbvCI nicking endonuclease at 37°C for 60 min;

[0057] Step 2: Add the reaction product of step 1 to a solution containing 200 μM signal probe, 1× The reaction buffer and 20 μL of a reaction solution containing 40 nM Tth Argonaute (TtAgo) protein were incubated at 75°C for 30 minutes before subsequent measurement.

[0058] Among them, piRNA is a ribonucleic acid that interacts with PIWI protein; the " / " in piRNA-36026 / piRNA-36743 represents one of or, and;

[0059] 3WJ means three-way connection;

[0060] KF polymerase is an N-terminal truncation of DNA polymerase I that retains DNA polymerase activity but loses 5′→3′ exonuclease activity;

[0061] Nt.BbvCI nicking endonuclease can specifically cut one of the strands on the double-stranded DNA substrate, and the resulting DNA molecule is "nicked" rather than cut;

[0062] NEBuffer 2 buffer is the reaction buffer that comes with the KF polymerase product;

[0063] rCutSmart buffer is the reaction buffer that comes with the Nt.BbvCI nicking endonuclease product;

[0064] The dNTP mixture is an equimolar solution of ultrapure dATP, dCTP, dGTP, and dTTP;

[0065] Tth Argonaute (TtAgo) is a DNA-guided endonuclease that cleaves the complementary sequence of the guide oligonucleotide. The cleavage site corresponds to a signal probe that is a DNA sequence modified with Cy5 or Cy3 at one end and a quencher group at the other end.

[0066] The reaction buffer is the reaction buffer that comes with the TthArgonaute protein product;

[0067] NEBuffer 2 buffer solution includes 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, and 1mM DTT.

[0068] rCutSmart buffer includes 500 mM potassium acetate, 200 mM triacetin, 100 mM magnesium acetate, 1 mg / ml recombinant albumin, pH 7.9.

[0069] The reaction buffer included Tris-HCl, (NH4)2SO4, KCl, MgSO4, and 1% poly(vinyl glycol)-p-isooctylphenyl ether.

[0070] The sequence of piRNA-36026 is: GGC CCCAUG GUG UAAUGG UCAGCACUC;

[0071] The sequence of piRNA-36743 is: GUU UCC GUAGUG UAG UGG UCAUCACGU UCG CC;

[0072] The sequence of 3WJ template-36026 is: ACA GGC CAG GAG CTG AGG AAC CAA GAG GCT GAG GATCTTATC CTT TTACAC CAT GGG G;

[0073] The sequence of 3WJ template-36743 is: CAACAAGAACTG CTGAGGAAAGTC TGT GCT GAG GAT CTTATCCTT CAC TACACTACG GA;

[0074] The sequence of 3WJ primer-36026 is: GAG TGC TGACCAGGATAA;

[0075] The sequence of 3WJ primer-36743 is: GCGAAC GTGATG CAG GATAA;

[0076] The sequence of linear template-36026 is: ACA GGC CAG GAG CTG AGG AAC CAA GAG GCT GA;

[0077] The sequence of linear template-36743 is: CAA CAA GAA CTG CTG AGG AAA GTC TGT GCT GA;

[0078] The sequence of signal probe-36026 is: Cy3-ACA GGC CAG GAG CTG A-BHQ2;

[0079] The sequence of signal probe-36743 is: Cy5-CAA CAA GAA CTG CTG A-BHQ3.

[0080] Example 2: The overall process is as follows Figure 1As shown. The present invention designs a three-way connection (3WJ) template, a 3WJ primer, a linear template and a signal probe for each piRNA. The 3WJ-36026 template consists of four characteristic regions: a partial complementary region of piR-36026, a hybridization region of 3WJ prime-36026, two recognition sites of Nt.BbvCI cutting endonuclease, and two SDA regions for generating trigger-36026 and gDNA 1. Similarly, the 3WJ template-36743 consists of four characteristic regions: a partial complementary region of piR-36743, a hybridization region of 3WJ primer-36743, two recognition sites of Nt.BbvCI cutting endonuclease, and two SDA regions for generating trigger-36743 and gDNA2. The 3WJ template with two T bases embedded in the middle region can form an unpaired nucleotide bulge at the 3WJ site to improve the efficiency of enzyme cutting. Linear template-36026 contains the binding region of trigger-36026, the recognition site of Nt.BbvCI, and the SDA region for generating gDNA 1. Linear template-36743 contains the binding region of trigger-36743, the recognition site of Nt.BbvCI, and the SDA region for generating gDNA 2. Reporter 1 and reporter 2 are modified with a fluorophore (Cy3 or Cy5) at the 5′ end and a quencher (BHQ2 or BHQ3) at the 3′ end, respectively, which can hybridize with gDNA 1 and gDNA 2, respectively, to initiate the TtAgo-dependent cyclic release of Cy3 and Cy5 fluorophores. This TtAgo biosensor includes three consecutive steps: (1) an enzyme cascade amplification reaction activated by target piRNAs, (2) TtAgo-dependent loop cleavage, and (3) single-molecule detection based on total internal reflection fluorescence (TIRF). In the presence of piR-36026, it can closely hybridize with 3WJ template-36026 and 3WJ primer-36026 to form a stable 3WJ structure. KlenowFragment extends the 3WJ primer in the stable 3WJ structure to generate a complete 3WJ structure with two Nt.BbvCI recognition sites. Nt.BbvCI then cuts the newly synthesized DNA chain on the 3WJ template, initiating circular strand displacement amplification (SDA) to produce a large number of amplicons with 3′-OH and 5′-PO4 ends (i.e., trigger-36026 and gDNA 1). The resulting trigger-36026 can serve as a primer paired with the linear template-36026, inducing continuous polymerization, nicking, and displacement to produce abundant gDNA 1 with a 5′-PO4 end. Similarly, in the presence of piR-36743, it can specifically hybridize with 3WJ template-36743 and 3WJ primer-36743 at a very close distance to form a stable 3WJ structure.Klenow fragment extends the 3WJ primer in the stable 3WJ structure to form a complete 3WJ structure with two Nt.BbvCI recognition sites. Nt.BbvCI then cuts the newly synthesized DNA chain on the 3WJ template, initiates circular SDA, and produces a large number of amplicons with 3′-OH and 5′-PO4 ends (i.e., trigger-36743 and gDNA2). The resulting trigger-36743 can serve as a primer paired with the linear template-36743 to induce continuous polymerization, nicking, and displacement to produce a large number of gDNA2 with 5′-PO4 ends. Ultimately, these gDNA 1 and gDNA 2 with 5′-PO4 ends can bind to TtAgo to form TtAgo / gDNA 1 and TtAgon / gDNA2 complexes, respectively, activating TtAgo-dependent recycling cleavage of reporter 1 and reporter 2, and releasing a large number of Cy3 and Cy5 fluorophores. Cy3 and Cy5 signals can be simply monitored by TIRF-based single-molecule imaging for the quantification of piR-36026 and piR-36743, respectively. In contrast, when piRNAs are absent, the enzymatic cascade cannot be initiated. As a result, no reporter probe can be cleaved and neither Cy3 nor Cy5 fluorescence signals can be detected.

[0081] Example 3: Feasibility of verification experiment

[0082] In order to study whether piR-36026 and piR-36743 can trigger enzyme cascade amplification, the present invention uses 14% non-denaturing PAGE with SYBR Gold as a fluorescent indicator to characterize the amplification products. When piR-36026 and piR-36743 are not present, no gDNA 1( Figure 2 A, lane 1) and gDNA2 ( Figure 2 B, lane 1), indicating that no enzymatic cascade amplification event occurred. However, when piR-36026 was present, a clear 16 nt band of the amplification product was observed ( Figure 2 A, lane 2), which is the same size as the synthetic gDNA 1 ( Figure 2 A, lane 3), indicating that a piR-36026-induced enzymatic cascade amplification event occurred. Similarly, the 16 nt band of the amplified product responded to piR-36743 ( Figure 2 B, lane 2), which matches the size of the synthetic gDNA2 ( Figure 2 Middle B, lane 3), indicating that piR-36744 can initiate an enzymatic cascade amplification event.

[0083] To further verify the feasibility of the constructed TtAgo biosensor, the present invention performed fluorescence emission spectroscopy measurements. In the presence of piR-36026, a high Cy3 fluorescence signal ( Figure 2 C, green line), high Cy5 fluorescence signal was detected in response to piR-36743 (see Figure 2 D, red line), indicating that piR-3606 and piR-36744 can activate the enzyme cascade amplification to produce abundant gDNA 1 and gDNA 2, respectively. The resulting gDNA 1 and gDNA 2 can hybridize with reporter 1 and reporter 2, respectively, to initiate the TtAgo-dependent cyclic release of Cy3 and Cy5 fluorescent molecules. In contrast, in the absence of piR-36026 and piR-36743, no obvious Cy3 ( Figure 2 C, gray line) and Cy5 fluorescence signal ( Figure 2 (D, blue line). Cy3 and Cy5 are respectively effective for piR-36026 ( Figure 2 Middle C, inset) and piR-36743 ( Figure 2 These results were also confirmed by the distinct green and red fluorescence images in middle D, inset. These results clearly demonstrated that this TtAgo biosensor can detect piR-36026 and piR-36743 simultaneously.

[0084] Example 4: Single-molecule detection of multiple piRNAs

[0085] The present invention uses TIRF for fluorescence imaging for single-molecule detection of multiple piRNAs. Figure 3 As shown, in the absence of piR-36026 and piR-36743, no Cy3 fluorescence signal could be detected ( Figure 3 A), and no Cy5 fluorescence signal could be detected ( Figure 3 E), indicating that no enzymatic cascade amplification occurred and therefore no cyclic release of Cy3 and Cy5 fluorescent molecules. In contrast, in the presence of piR-36026, only Cy3 fluorescent signal was generated ( Figure 3 B), but no Cy5 fluorescence signal was detected ( Figure 3 In the presence of piR-36743, only Cy5 fluorescence signal was generated ( Figure 3 G), but no Cy3 fluorescence signal was detected ( Figure 3 C), indicating that piR-36026 and piR-36744 can activate the enzyme cascade amplification, thereby initiating the subsequent cyclic release of Cy3 and Cy5 molecules, respectively. When both piR-36026 and piR-36743 are present, Cy3 ( Figure 3 D) and Cy5( Figure 3H) Fluorescence signal. It is worth noting that the Cy3 fluorescent spots in the single-molecule fluorescence image show a one-step photobleaching ( Figure 3 I), indicating that the detected spots represent single Cy3 molecules. The Cy5 fluorescent spots in the single-molecule fluorescent image also show a step of photobleaching ( Figure 3 J), indicating that the detected spots represent single Cy5 molecules. All the above results confirm that this TtAgo biosensor allows the simultaneous detection of multiple piRNAs at the single-molecule level.

[0086] Example 5: Sensitivity Analysis

[0087] like Figure 4 As shown in A and B, the Cy3 and Cy5 counts (N) increased with the concentration of piR-36026 and piR-36743 (C) from 0 to 1×10 -8 On a logarithmic scale, the counts of Cy3 and Cy5 were correlated with the concentration of piR-36026 and piR-36743 at 1×10 -16 Up to 1×10 -10 There is a linear correlation over the six orders of magnitude of M ( Figure 4 C and D). The regression equations of piR-36026 and piR-36743 were N = 696.13 + 40.21lgC (R 2 =0.996) and N = 699.78 + 39.14lgC (R 2 =0.990). The limits of detection (LOD) of piR-36026 were calculated to be 10.23 aM and 5.89 aM, respectively. The ultrahigh sensitivity of this TtAgo biosensor can be attributed to the following factors: (1) the high efficiency of enzyme cascade amplification, (2) the precise recognition and high cleavage activity of TtAgo, and (3) the high signal-to-noise ratio of single-molecule detection.

[0088] Example 6: Specificity analysis

[0089] The selectivity of this TtAgo biosensor was evaluated by introducing several non-target RNAs (i.e., circMTO1, piR-651, piR-932, DSCAM-AS1, miR-10b, and miR-210) as interfering RNAs. In theory, none of these interfering RNAs can activate the enzyme cascade amplification event and therefore will not release Cy3 and Cy5 fluorophores. The present invention compared the Cy3 and Cy5 fluorescence signals generated by piR-36026 and piR-36743, respectively, with those generated by the interfering RNAs. Figure 5As shown, these interfering RNAs (circMTO1, piR-651, piR-932, DSCAM-AS1, miR-10b, and miR-210) could neither obtain Cy3 nor Cy5 fluorescence signals, which was similar to the control group without any RNA. In contrast, the presence of piR-36026 could induce a significant Cy3 fluorescence signal, while the presence of piR-36743 could produce a significant Cy5 fluorescence message. The presence of piR-36726 and piR-36744 could simultaneously obtain different Cy3 and Cy5 fluorescence signals, indicating that only piR-3606 and piR-3.6743 could activate the enzyme cascade amplification to initiate the cyclic release of Cy3 and Cyt5 fluorophores. These results were further confirmed by the radar plot, which provided a visual comparison between several quantitative aspects of the situation. Therefore, this TtAgo biosensor has good selectivity for piR-36026 and piR-36743 and has potential application prospects in complex biological environments.

[0090] Example 7: Actual sample analysis

[0091] The cellular piRNAs sensing ability of this TtAgo biosensor was explored in five cells with different piR-36026 and piR-36743 expression levels, including BT474 cells, HeLa cells, A549 cells, MCF-7 cells, and MCF-10A cells ( Figure 6 According to A). Figure 3 The concentrations of piR-36026 and piR-36743 were calculated using the calibration curves in Figure 1. Compared with the negligible signal in normal cells (i.e., MCF-10A cells), high expression levels of piR-36026 were observed in four cancer cells (MCF-7 cells, BT474 cells, HeLa cells, and A549 cells). Figure 6 In B, green column). In addition, piR-36743 ( Figure 6 The expression level of (C, red column) in MCF-10A cells was much lower than that in the four cancer cells. The present invention used the same batch of extract samples from BT474 cells, HeLa cells, A549 cells, MCF-7 cells and MCF-10A cells to perform standard qRT-PCR ( Figure 6 The results of the present invention are consistent with those obtained by qRT-PCR ( Figure 6 B and C), demonstrating the feasibility and accuracy of this TtAgo biosensor for real sample analysis. It is noteworthy that there is a good linear relationship between Cy3 counts (N) and the logarithm of the MCF-7 cell number (X) in the range of 1 to 100,000 cells ( Figure 6The regression equation is N = 85.26 + 34.85lgX, and the correlation coefficient is 0.999. The detection limit is directly measured as 1 cell. Similarly, the Cy5 count (N) is linearly related to the logarithm of the BT474 cell number (X) from 1 to 100,000 cells ( Figure 6 E). The regression equation is N = 118.64 + 32.66lgX (R 2 =0.999), with a detection limit of 1 cell. These results indicate that the constructed TtAgo biosensor can be used to accurately quantify multiple piRNAs at the single-cell level.

[0092] Example 8: Clinical sample analysis

[0093] To confirm the potential of this TtAgo biosensor in clinical diagnosis, the present invention measured the expression levels of piR-36026 and piR-36743 in tissue samples from cancer patients (n=11) and healthy donors (n=5). The concentration of piR-36026 in breast cancer patient tissues was significantly higher than that in healthy donor tissues ( Figure 7 The median concentration in cancer patients was 27.7 pM, and that in healthy donors was 4.3 pM ( Figure 7 In addition, the concentration of piR-36743 in breast cancer patient tissues was much higher than that in healthy donor tissues ( Figure 7 C), the median concentration in cancer patients was 14.6 pM, and that in healthy donors was 4.2 pM ( Figure 7 The results obtained by the proposed TtAgo biosensor were in good agreement with those obtained by qRT-PCR for each sample ( Figure 7 These results suggest that piR-36026 and piR-36743 can be used as tumor biomarkers for early diagnosis of cancer.

[0094] The Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification of the present invention and its preparation method and use are used for simultaneously detecting multiple piRNAs. Compared with existing piRNA detection methods, the biosensor has significant advantages: this TtAgo biosensor has significant advantages such as simple design, high amplification efficiency and flux, and good specificity. This TtAgo biosensor can greatly increase the amount of gDNA through enzyme cascade amplification, which helps to release abundant Cy3 / Cy5 molecules to produce enhanced fluorescence signals. Compared with the classic EXPER using SYBR dyes that can only detect a single target RNA, this TtAgo biosensor can detect multiple piRNAs simultaneously. CRISPR / Cas nucleases involve long and easily degradable RNA guides, while TtAgo only requires 16nt DNA guides, thereby reducing costs and improving detection stability. This TtAgo biosensor can easily detect any RNA target of interest because it is not limited by sequences such as PAM motifs, increasing its versatility in diagnostic applications. By cleverly combining the unique features of TtAgo, such as efficient enzyme cascade amplification, programmability, and multi-flip cleavage activity, as well as the high signal-to-noise ratio of single-molecule detection, the TtAgo biosensor exhibits excellent specificity and high sensitivity over a large dynamic range of 6 orders of magnitude, with a detection limit of 8.51aM for piR-36026 and 13.34aM for piR-36743. In addition, the TtAgo biosensor can distinguish cancer cells and normal cells with high expression of piRNAs, and simultaneously quantify multiple piRNAs in cancer cells at the single-cell level, and can be further used to accurately distinguish cancer patients from healthy donors.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification, characterized in that: The following steps are involved: Step 1, incubate a reaction solution containing piRNA-36026 / piRNA-36743, 3WJ template, 3WJ primer, linear template, NEBuffer2 buffer, rCutSmart buffer, dNTPs mixed solution, KF polymerase, and Nt.BbvCI nicking endonuclease; Among them, piRNA is a ribonucleic acid that interacts with PIWI protein; the " / " in piRNA-36026 / piRNA-36743 represents one of or, and; 3WJ means three-way connection; KF polymerase is an N-terminal truncation of DNA polymerase I that retains DNA polymerase activity but loses 5′→3′ exonuclease activity; Nt.BbvCI nicking endonuclease can specifically cut one of the strands on the double-stranded DNA substrate, and the resulting DNA molecule is "nicked" rather than cut; NEBuffer 2 buffer is the reaction buffer that comes with the KF polymerase product; rCutSmart buffer is the reaction buffer that comes with the Nt.BbvCI nicking endonuclease product; The dNTP mixture is an equimolar solution of ultrapure dATP, dCTP, dGTP, and dTTP; Step 2, adding the reaction product of step 1 to a Incubate in a reaction buffer and a reaction solution of TthArgonaute protein to obtain a Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification; Among them, Tth Argonaute is a DNA-guided nuclease that cuts the complementary sequence of the guide oligonucleotide. The cleavage site corresponds to a signal probe that is a DNA sequence modified with Cy5 or Cy3 at one end and a quenching group at the other end. The reaction buffer is the reaction buffer that comes with the TthArgonaute protein product.

2. The method for preparing the Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification according to claim 1, characterized in that: In the step 1, 10 μL of a reaction solution containing different concentrations of piRNA-36026 / piRNA-36743, 100 nM 3WJ template, 100 nM 3WJ primer, 100 nM linear template, 1×NEBuffer 2, 1×rCutSmart buffer, 200 μM dNTPs mixed solution, 2 U KF polymerase, and 8 U Nt.BbvCI nicking endonuclease was incubated at 37° C. for 60 minutes.

3. The method for preparing the Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification according to claim 1, characterized in that: In step 2, the reaction product is added to a solution containing 200 μM signal probe, The cells were incubated at 75°C for 30 minutes in a reaction buffer and 20 μL of a reaction solution containing 40 nM Tth Argonaute protein.

4. The method for preparing the Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification according to claim 1, characterized in that: The NEBuffer 2 buffer solution includes 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM DTT.

5. The method for preparing the Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification according to claim 1, characterized in that: The rCutSmart buffer includes 500 mM potassium acetate, 200 mM triacetin, 100 mM magnesium acetate, 1 mg / ml recombinant albumin, and has a pH of 7.

9.

6. The method for preparing the Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification according to claim 1, characterized in that: Said The reaction buffer included Tris-HCl, (NH4)2SO4, KCl, MgSO4, and 1% polyoxyethylene glycol p-isooctylphenyl ether.

7. The method for preparing the Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification according to claim 1, characterized in that: The sequence of piRNA-36026 is: GGC CCC AUG GUG UAA UGG UCA GCA CUC; The sequence of piRNA-36743 is: GUU UCC GUA GUG UAG UGG UCA UCA CGU UCG CC; The 3WJ templates include 3WJ template-36026 and 3WJ template-36743. The sequence of the 3WJ template-36026 is: ACA GGC CAG GAG CTG AGG AAC CAA GAG GCT GAG GATCTT ATC CTT TTA CAC CAT GGG G; The sequence of the 3WJ template-36743 is: CAA CAA GAA CTG CTG AGG AAA GTC TGT GCT GAG GATCTT ATC CTT CAC TAC ACT ACG GA; The 3WJ primers include 3WJ primer-36026 and 3WJ primer-36743, The sequence of the 3WJ primer-36026 is: GAG TGC TGA CCA GGA TAA; The sequence of the 3WJ primer-36743 is: GCG AAC GTG ATG CAG GAT AA; The linear templates include linear template-36026 and linear template-36743, The linear template-36026 sequence is: ACA GGC CAG GAG CTG AGG AAC CAA GAG GCT GA; The linear template -36743 sequence is: CAA CAA GAA CTG CTG AGG AAA GTC TGT GCT GA; The signal probes include signal probe-36026 and signal probe-36743, The signal probe-36026 sequence is: Cy3-ACA GGC CAG GAG CTG A-BHQ2; The sequence of the signal probe-36743 is: Cy5-CAA CAA GAA CTG CTG A-BHQ3.

8. An enzyme cascade amplification-regulated Thermus thermophilus Argonaute biosensor obtained by the preparation method of claim 1.

9. Use of the Thermus thermophilus Argonaute biosensor regulated by enzyme cascade amplification according to claim 8 as a biosensor for simultaneously detecting multiple piRNAs.

10. The use according to claim 9, characterized in that: The detection limits of the biosensor for piR-36026 were 8.51 aM and that for piR-36743 was 13.34 aM.

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