Exponential rolling circle amplification method based on Argonaute protein

Abstract

The invention provides an exponential rolling circle amplification method based on Argonaute protein. The exponential rolling circle amplification method comprises the following steps: providing a 5'end phosphorylated single-chain gDNA; the method comprises the following steps: carrying out hybridization annealing on target RNA (Ribonucleic Acid) and a Padlock probe to form an RNA-Padlock compound; under the action of ligase, cyclizing the RNA-Padlock compound to form a circular DNA (Deoxyribose Nucleic Acid) template; the gDNA and TtAgo are subjected to pre-incubation, and a TtAgo / gDNA compound is formed; the method comprises the following steps: mixing a circular DNA template, a TtAgo / gDNA compound, DNA polymerase, dNTPs and a fluorescent dye in a same reaction system, and carrying out an exponential rolling circle amplification reaction. According to the exponential rolling circle amplification method based on the Argonaute protein, accurate shearing can be achieved at any preset position of an RCA product, a specific enzyme cutting site does not need to be introduced, system construction is greatly simplified, and exponential amplification is achieved.

Description

Technical Field

[0001] This invention relates to an exponential rolling circle amplification method based on Argonaute protein, belonging to the field of biotechnology. Background Technology

[0002] MicroRNAs (miRNAs) are a class of non-coding small RNA molecules, approximately 18–25 nt in length, that are widely involved in gene expression regulation and exhibit abnormal expression characteristics closely related to the occurrence and development of various pathological processes, including tumors, cardiovascular diseases, and neurodegenerative diseases. Therefore, the sensitive, specific, and convenient detection of miRNAs is of great significance in early disease diagnosis, prognostic assessment, and personalized medicine. However, due to their small molecular weight, highly similar sequences, and extremely low abundance in biological samples, their detection has always been a focus in the field of nucleic acid analysis.

[0003] Nucleic acid amplification technology is a crucial foundation for molecular diagnostics and bioassay. Among these methods, rolling circle amplification (RCA) is a classic isothermal nucleic acid amplification technique. The reaction relies on DNA polymerase to continuously replicate on a circular template (CT), generating ultra-long single-stranded DNA products. Due to its lack of the need for thermal cycling, ease of operation, and diverse product forms, RCA has been widely used in nucleic acid detection, molecular diagnostics, signal amplification, and nanobiology since its inception.

[0004] The earliest form of RCA was linear RCA, which uses only one primer for continuous extension on a CT scanner. In linear RCA, each circular template can only induce the replication of a single strand, and the product accumulation follows a linear pattern. The detection sensitivity is usually low, making it difficult to meet the high-sensitivity detection requirements for low-abundance biomarkers, especially short nucleic acid fragments such as microRNA, in clinical or field settings.

[0005] To overcome the problem of insufficient sensitivity, the academic community has proposed several improvement strategies, as follows:

[0006] 1. Multiprimed RCA (mRCA) involves annealing multiple primers simultaneously on a larger circular template, which enables the polymerization reaction to be initiated at multiple sites at the same time, significantly increasing the product accumulation rate. This method has application value in genome sequencing and copy amplification. Its detection sensitivity is improved compared to linear RCA, but its applicability to short targets such as microRNA is limited.

[0007] 2. Hyperbranched RCA (HRCA) involves introducing a second primer onto the initial product chain, utilizing its complementary region to anneal and extend the chain, thereby continuously triggering chain substitution reactions. This transforms the amplification process from an arithmetic mode to an exponential mode, significantly improving detection sensitivity.

[0008] 3. Circle-to-circle amplification (C2CA) involves digesting the initial RCA product with restriction endonucleases and recirculating it to generate a new circular template for the next round of RCA, thus creating a cyclic amplification. C2CA further amplifies the signal, and reports indicate it can detect as few as hundreds to thousands of copies of nucleic acid. However, this method requires additional enzyme digestion and circularization steps, making it cumbersome and complex, which hinders its widespread application.

[0009] 4. Primer-generated RCA (PG-RCA) continuously generates new 3′ ends through nicking enzymes, triggering subsequent extension reactions and thus achieving higher sensitivity. However, both C2CA and PG-RCA depend on exogenous enzyme digestion steps, and the nicking enzymes are sequence-dependent, making their design complex.

[0010] Overall, the aforementioned improved strategies such as multi-primer RCA (mRCA), superbranched RCA (HRCA), circle-to-circle amplification (C2CA), and primer-generated RCA (PG-RCA) have improved amplification rate and sensitivity to some extent, but the following problems still exist: First, they generally rely on exogenous primers or specific enzyme digestion sequences, which increases design complexity and brings potential risks of non-specific amplification; second, the amplification efficiency is still limited, making it difficult to meet the high-sensitivity detection requirements of trace nucleic acids such as low-abundance miRNAs.

[0011] Therefore, it is necessary to design a new exponential rolling circle amplification method based on Argonaute protein to overcome the above problems. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and provide an exponential rolling circle amplification method based on Argonaute protein, which can achieve precise cleavage at any preset position of RCA product without the need to introduce specific enzyme cleavage sites, greatly simplifying system construction and realizing exponential amplification.

[0013] This invention is implemented as follows:

[0014] This invention provides an exponential rolling circle amplification method based on Argonaute protein, comprising the following steps:

[0015] Step 1: Provide a 5' phosphorylated single-stranded gDNA, wherein the gDNA is capable of specifically targeting different predetermined positions on a circular DNA template;

[0016] Step 2: Hybridize and anneal the target RNA with the first-line Padlock probe to form an RNA-Padlock complex;

[0017] Step 3: Under the action of ligase, the RNA-Padlock complex prepared in step 2 is circularized to form a circular DNA template;

[0018] Step 4: Pre-incubate the gDNA from Step 1 with TtAgo in the presence of magnesium ions to form a TtAgo / gDNA complex with targeted cleavage activity.

[0019] Step 5: The circular DNA template, the TtAgo / gDNA complex, a DNA polymerase with strand displacement activity, dNTPs, and a fluorescent dye are mixed in the same reaction system to perform an exponential rolling circle amplification reaction. The TtAgo / gDNA complex specifically cleaves the circular DNA template to initiate rolling circle amplification, and the amplified repetitive units containing the target sequence can be cleaved again by the TtAgo / gDNA complex, continuously generating new replication origins and achieving exponential amplification.

[0020] Furthermore, in step two, the annealing conditions are: annealing at 85°C and an annealing rate of 1°C / minute.

[0021] Furthermore, in step three, the ligase is Splint R ligase.

[0022] Furthermore, in step three, Splint R reaction buffer and RNase inhibitor were added during the cyclization process.

[0023] Furthermore, in step four, the pre-incubation temperature is 70-80℃, and the incubation time is 20-30 minutes.

[0024] Furthermore, in step five, the DNA polymerase with strand displacement activity is Vent DNA polymerase.

[0025] Furthermore, in step five, the fluorescent dye is SYBR Green I.

[0026] Furthermore, in step five, the temperature cycling of the exponential rolling circle amplification reaction is 55°C for 30 seconds and 85°C for 30 seconds, with a cycle count of 90-150 times.

[0027] Furthermore, in step five, the generation of exponential rolling circle amplification products is detected by real-time monitoring of the signal changes of the fluorescent dye, thereby enabling the detection of the target RNA.

[0028] The present invention has the following beneficial effects:

[0029] The exponential rolling circle amplification method based on Argonaute protein provided by this invention utilizes the gDNA-guided cleavage mechanism of TtAgo to achieve precise cleavage at any preset location in the RCA product without the need for specific restriction enzyme sites. This greatly simplifies system construction and enables exponential amplification. Real-time fluorescence monitoring results show that this system can achieve highly efficient exponential amplification of miRNAs, significantly improving detection sensitivity and reaction efficiency while maintaining system simplicity, providing a new solution for the detection of low-abundance nucleic acid biomarkers. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 A schematic diagram illustrating the principle of the exponential rolling circle amplification method based on Argonaute protein provided in this embodiment of the invention;

[0032] Figure 2 A schematic diagram of the real-time fluorescence quantitative results of three different VeAgo gDNAs provided in an embodiment of the present invention;

[0033] Figure 3 A schematic diagram of the 2% agarose gel electrophoresis results of three different VeAgo gDNAs provided in an embodiment of the present invention;

[0034] Figure 4 This is a schematic diagram of the sensitivity analysis of VeAgo provided in an embodiment of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] like Figure 1As shown, this invention provides an exponential rolling circle amplification method based on the Argonaute protein, namely, a primer-free exponential amplification system driven by TtAgo (named VeAgo). This system uses miRNA as a target to guide the Padlock probe to circularize into a circular template (CT). During the RCA reaction, pre-designed gDNA guides TtAgo to specifically cleave the amplification product, thereby continuously generating new primer binding sites on the synthetic strand using Vent DNA polymerase. The released fragments can be directly used as functional primers to re-enter the amplification cycle, forming a self-feedback amplification mechanism that couples cleavage and extension, transforming the RCA from a linear mode to an exponential mode. TtAgo (Thermus thermophilus Argonaute) is a prokaryotic Argonaute protein derived from Gram-negative thermophilic bacteria. It achieves precise guidance of single-stranded guide DNA (gDNA) through 5' phosphorylation, enabling DNA-guided specific nucleic acid cleavage under high-temperature conditions of 65–85 °C. Unlike nickases that rely on fixed recognition sequences, TtAgo can precisely cleave target DNA using only a short 5′ phosphorylated guide DNA (gDNA). It offers flexible recognition range and controllable targeting, eliminating the need to artificially introduce specific sequences into the template and greatly expanding the freedom of system design.

[0037] This invention provides an exponential rolling circle amplification method based on Argonaute protein, comprising the following steps:

[0038] Step 1: Provide a 5' phosphorylated single-stranded gDNA, wherein the gDNA is capable of specifically targeting different predetermined positions on a circular DNA template.

[0039] Step 2: Hybridize and anneal the target RNA with the first-line Padlock probe to form an RNA-Padlock complex; the annealing conditions are: annealing at 85°C and an annealing rate of 1°C / min.

[0040] Step 3: The RNA-Padlock complex prepared in Step 2 is circularized under the action of a ligase to form a circular DNA template; wherein the ligase is Splint R ligase; Splint R reaction buffer and RNase inhibitor are added during the above circularization process.

[0041] Step 4: Pre-incubate the gDNA from Step 1 with TtAgo in the presence of magnesium ions to form a TtAgo / gDNA complex with targeted cleavage activity; wherein the pre-incubation temperature is 70-80℃ and the incubation time is 20-30 minutes.

[0042] Step 5: The circular DNA template, the TtAgo / gDNA complex, a DNA polymerase with strand displacement activity, dNTPs, and a fluorescent dye are mixed in the same reaction system to perform an exponential rolling circle amplification reaction. The TtAgo / gDNA complex specifically cleaves the circular DNA template to initiate rolling circle amplification, and the amplified repetitive units containing the target sequence can be further cleaved by the TtAgo / gDNA complex, continuously generating new replication origins, thus achieving exponential amplification. The DNA polymerase with strand displacement activity is Vent DNA polymerase; the fluorescent dye is SYBR Green I. The temperature cycling for the exponential rolling circle amplification reaction is 55°C for 30 seconds and 85°C for 30 seconds, with 90-150 cycles. Furthermore, the generation of the exponential rolling circle amplification product is detected by real-time monitoring of the fluorescent dye signal changes, thereby enabling the detection of the target RNA.

[0043] The following specific examples illustrate this, where the concentrations of each substance are expressed as (initial concentration and final concentration):

[0044] Step 1: Design and preparation of guide DNA (gDNA).

[0045] To ensure specific target recognition and precise localization, single-stranded gDNA with 5' phosphorylation modification was designed. To investigate the effects of three different cleavage sites on the amplification reaction, three different gDNA sequences were designed, located at the front, middle, and back ends of the circular product, respectively, to generate different cleavage sites for comparison.

[0046] Step 2: Annealing of the target RNA with Padlock (15 μL)

[0047] Reactants:

[0048] MgCl2 (magnesium chloride): provides Mg²⁺ ions, which are essential ions for RNA annealing reactions.

[0049] Let-7a RNA: Target RNA, a type of microRNA.

[0050] Padlock-P (lock probe): is a linear probe that pairs complementary to let-7a RNA head-to-tail. After annealing, it can pair with the target RNA and form a circular structure.

[0051] Test Control <![CDATA[H2O]]> 0 6 μL <![CDATA[MgCl2(10 mM→2 mM)]]> 3 μL 3 μL Let-7a RNA (10 µM → 4 µM) 6 μL 0 Padlock-P (10 µM → 4 µM) 6 μL 6 μL

[0052] Operate on the ice box.

[0053] Reaction conditions: RNA annealing (85℃, 1℃ / min) to obtain padlock (4 µM).

[0054] Process description:

[0055] Annealing conditions: Annealing at 85°C at a rate of 1°C / min, ultimately forming a stable RNA-Padlock complex.

[0056] The product obtained: After annealing, a stable RNA-Padlock complex was formed at a concentration of 4 µM.

[0057] Step 3: Circulation of the Padlock probe (10 µL), product name: CT

[0058] Reactants:

[0059] 10× Splint R reaction buffer: A buffer solution for using ligases to provide the buffer environment required for the reaction.

[0060] RNase inhibitors: prevent RNA degradation.

[0061] Splint R ligase (Splint R): A ligase specifically designed to close circular probes, connecting the gaps in the circularization of padlock probes.

[0062] Circular probe (Padlock): at a concentration of 4 µM, it will undergo a cyclization reaction.

[0063] Name (Name) Volume(μL) <![CDATA[DEPC H2O]]> 2 μL 10×splint R reaction buffer 1 μL RNase inhibitor(10 U / µL → 1 U / µL) 1 μL Splint R (10 U / µL → 1 U / µL) 1 μL Padlock (4 µM → 2 µM) 5 μL

[0064] Reaction conditions: 25 ℃ for 1 h

[0065] Process description:

[0066] Circulation reaction: Catalyzed by Splint R ligase, the gaps in the padlock probe circularization are joined together and closed to form a circular DNA template, called CT.

[0067] Step 4: Incubation with TtAgo / gDNA (20 µL total for steps 4 and 5)

[0068] Reactants:

[0069] 10× Thermol buffer: Provides a suitable reaction environment for TtAgo (Tth Argonaute).

[0070] MgSO4 (magnesium sulfate): regulates magnesium ion concentration and promotes enzyme activity.

[0071] gDNA: guides TtAgo to undergo a cleavage reaction.

[0072] TtAgo: Thermus thermophilus argonaute (TtAgo).

[0073] Name (Name) Volume(μL) <![CDATA[H2O]]> 4 μL 10× Thermol buffer 2 μL <![CDATA[MgSO4(60 mM-6 mM)]]> 2 μL gDNA (5 µM - 250 nM) / gDNA-1, gDNA-2, gDNA-3 1 μL TtAgo (1 µM → 50 nM) 1 μL

[0074] Reaction conditions: 75 ℃ for 25 min

[0075] Process description:

[0076] Incubation reaction: TtAgo is mixed with gDNA and incubated to allow it to bind in advance.

[0077] Incubation conditions: Temperature set at 75°C, incubation time 25 minutes.

[0078] Step 5: VeAgo (for RCA amplification and SYBR Green I dye detection)

[0079] Reactants:

[0080] 10× Thermol buffer: Provides a buffer environment for the VeAgo protocol.

[0081] DTT (dithiothreitol): used to reduce any disulfide bonds that may exist, thus maintaining protein activity.

[0082] dNTPs (deoxyribonucleoside triphosphates): provide the basic materials needed for DNA synthesis.

[0083] CT (Circularized Probe): The circularized product obtained from step three will be used for amplification.

[0084] Vent enzyme: a thermostable DNA polymerase used for DNA amplification.

[0085] 20×SYBR Green I: A fluorescent dye with high affinity for double-stranded DNA. Its staining principle is through intercalation or binding in the minor groove of the DNA double strand. When SYBR Green I binds to single-stranded DNA or free nucleotides, the fluorescence signal is weak, but when it binds to double-stranded DNA, the fluorescence intensity is significantly enhanced, emitting a strong green fluorescence upon excitation.

[0086] Name (Name) Volume(μL) 10×Thermol buffer 2 μL DTT (10 mM → 0.5 mM ) 1 μL dNTPs(25 mM → 1.25mM) 1 μL CT(2 µM → 800 nM) 2.5 μL Vent (2 U / µL → 0.25U / µL) 2.5 μL 20×SYBR green i 1 μL Ago / gDNA mix 10 μL

[0087] Reaction conditions: 55 ℃ for 30 s, 85 ℃ for 30 s, 120 cycles in a real-time fluorescence quantitative PCR instrument.

[0088] Process description:

[0089] Amplification and detection: Amplification reaction was performed using Vent DNA polymerase, and the amplification products were detected using SYBR Green I dye.

[0090] Amplification conditions: The reaction temperature was set at 55°C (30 seconds) and 85°C (30 seconds) for 120 cycles.

[0091] The product obtained: The product amplified by the VeAgo protocol can be verified by monitoring the amplification curve dynamically with SYBR Green I to confirm the exponential amplification of the product.

[0092] The exponential amplification of the above products was verified using multiple detection methods:

[0093] Figure 2 Real-time fluorescence quantitative detection results for three different VeAgo gDNAs:

[0094] Results Analysis: Real-time fluorescence quantitative kinetic curves showed that all three gDNAs in the experimental group exhibited a rapid increase in fluorescence signal at the initial stage of the reaction, and entered a plateau phase after approximately 40 cycles. In contrast, the three gDNAs in the control group did not show significant signals within the same number of cycles, only gradually increasing in intensity after approximately 100 cycles, with a significantly delayed rate of signal increase. Among the three experimental gDNAs, gDNA2 exhibited the highest fluorescence intensity, a stable plateau phase, the lowest background, and the best signal-to-noise ratio.

[0095] These results demonstrate that the VeAgo protocol can effectively initiate the amplification reaction in the presence of the target, validating its feasibility. Furthermore, gDNA2 exhibits the best detection performance, demonstrating higher fluorescence signal and lower background interference, thus exhibiting the best sensitivity and reliability. Therefore, gDNA2 was selected for real-time quantitative PCR in subsequent sensitivity analysis.

[0096] Figure 3 Results of 2% agarose gel electrophoresis of three different VeAgo gDNAs:

[0097] Results analysis: 2% agarose gel electrophoresis showed that clear bands appeared in experimental groups T1, T2, and T3, indicating successful amplification product generation; among them, the T2 band was the clearest, brightest, and had the best amplification efficiency. In contrast, no clear bands were detected in the control groups C1, C2, and C3, indicating that non-specific amplification would not occur under target-free conditions.

[0098] The results indicate that the VeAgo protocol can effectively generate amplification products in the presence of the target, verifying the feasibility of the protocol. Furthermore, the T2 amplification effect is the best, which is consistent with the results of real-time fluorescence quantification.

[0099] Figure 4 Sensitivity analysis for VeAgo:

[0100] Results Analysis: Real-time quantitative PCR sensitivity analysis showed that different target concentrations could induce amplification reactions, and the higher the target concentration, the earlier the curve appeared. High-concentration samples (10 nM, 5 nM) entered the exponential amplification phase in about 20 cycles and quickly reached a stable plateau; medium-concentration samples (1 nM, 500 pM) showed obvious amplification signals in about 30–40 cycles, and the curve morphology was clear and distinguishable; low-concentration samples (100 pM) only began to show fluorescence enhancement after about 60 cycles, and the amplification curve was still identifiable, but the signal was relatively weak. When the concentration was further reduced to 10 pM or 1 pM, the amplification signal showed a significant delay and was close to that of the blank control group, making it difficult to reliably distinguish.

[0101] The results show that the detection sensitivity of this scheme is between 100 pM and 500 pM, and a reliable detection signal can still be obtained within this concentration range, verifying the feasibility of the scheme in the detection of low-concentration targets.

[0102] sequence:

[0103] Name Sequence (5’-3’) Padlock-P G CTA CTA CCG AGT CAT TTG CCC TTT ACA TTT CAC TTT CCC TTC ACC TTT AAC TAT ACA AC Let-7a UG AGG UAG UAG GUU GUA UAG UU gDNA-1 TACTACCGAGTCATTTG gDNA-2 TTTACATTTCACTTTCC gDNA-3 TTTCCCTTCACCTTTAA

[0104] All sequences used were synthesized at Shanghai Sangon Biotech Co., Ltd. gDNA was phosphorylated at the 5' end, purified by HPLC and verified by mass spectrometry, and stored at -20℃ for later use. Tth Argonaute (TtAgo), Vent DNA Polymerase (exo-), Splint R, RNase inhibitor, dNTPs, DTT, and SYBR green i were all purchased from NEB. The buffers used were NEB's by-products. The 1× ThermoPol Reaction Buffer contained: 20 mM Tris-HCl, 10 mM KCl, 10 mM NH4)2SO4, 2 mM MgSO4, and 0.1% Triton X-100, with a pH of 8.8. The 1× splint R reaction buffer contained: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, with a pH of 7.5 and a storage temperature of 25℃.

[0105] In summary, the present invention has the following beneficial effects:

[0106] 1. The exponential rolling circle amplification method based on Argonaute protein provided by this invention utilizes the gDNA-guided cleavage mechanism of TtAgo to achieve precise cleavage at any preset position of the RCA product without the need to introduce specific restriction enzyme sites, thus greatly simplifying system construction and achieving exponential amplification. Real-time fluorescence monitoring results show that this system can achieve highly efficient exponential amplification of miRNAs, significantly improving detection sensitivity and reaction efficiency while maintaining system simplicity, providing a new solution for the detection of low-abundance nucleic acid biomarkers.

[0107] 2. Achieving in vivo regeneration and continuous supply of primers (primer recycling). Relying on gDNA-guided TtAgo to perform site-specific cleavage on the amplification strand, the released oligonucleotide fragments can be used as new initiating primers to repeatedly participate in amplification, forming a self-sustaining positive feedback loop of "shearing → primer generation → extension → re-shearing", thereby enabling the system to have a continuous primer supply capacity.

[0108] 3. Transforming linear or weak exponential amplification into more stable exponential amplification. As primers are continuously regenerated during the reaction and used at multiple sites, the amplification kinetics shift from linear accumulation with a single template to exponential growth with self-feedback, significantly increasing the product accumulation rate. This helps to shorten detection time and improve the detection capability of low-copy targets.

[0109] 4. No need to embed fixed restriction enzyme recognition sequences in the template, allowing for greater design flexibility. Unlike C2CA and PG-RCA, which rely on restriction / cleavage enzymes, the cleavage site of TtAgo is specified by a synthetically produced short-chain gDNA, thus eliminating the need to pre-install specific restriction sequences in padlock or CT, resulting in greater design freedom.

[0110] 5. Short gDNA is low-cost and easy to design / synthesize. The gDNA required for TtAgo is usually a short oligonucleotide of 15–20 nt. Its synthesis cost and quality control are more economical and convenient than long guide RNA or multiple long primers, which facilitates batch preparation and rapid iterative optimization.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An exponential rolling circle amplification method based on Argonaute proteins, characterized in that, The method comprises the following steps: Step 1: providing a 5'-phosphorylated single-stranded gDNA, which can specifically target different predetermined positions of a circular DNA template; Step 2: hybridizing and annealing a target RNA with a linear Padlock probe to form an RNA-Padlock complex; Step 3: circularizing the RNA-Padlock complex prepared in step 2 under the action of a ligase to form a circular DNA template; Step 4: pre-incubating the gDNA in step 1 with TtAgo in the presence of magnesium ions to form a TtAgo / gDNA complex with targeted cleavage activity; Step 5: mixing the circular DNA template, the TtAgo / gDNA complex, a DNA polymerase with strand displacement activity, dNTPs and a fluorescent dye in the same reaction system to perform an exponential rolling circle amplification reaction; wherein the specific cleavage of the TtAgo / gDNA complex to the circular DNA template initiates the rolling circle amplification, and the repeated units containing the target sequence generated by the amplification can be cleaved again by the TtAgo / gDNA complex to continuously generate new replication origins, thereby realizing exponential amplification.

2. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 2, the annealing conditions are: annealing at 85°C with an annealing rate of 1°C / min.

3. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 3, the ligase is a Splint R ligase.

4. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 3, Splint R reaction buffer and RNase inhibitor are added during the circularization process.

5. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 4, the pre-incubation temperature is 70-80°C, and the incubation time is 20-30 minutes.

6. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 5, the DNA polymerase with strand displacement activity is Vent DNA polymerase.

7. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 5, the fluorescent dye is SYBR Green I.

8. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 5, the temperature cycle of the exponential rolling circle amplification reaction is 55°C for 30 seconds and 85°C for 30 seconds, and the cycle number is 90-150 times.

9. The Argonaute protein-based exponential rolling circle amplification method of claim 1, wherein: In step 5, the generation of the exponential rolling circle amplification product is detected by real-time monitoring of the signal change of the fluorescent dye, so as to realize the detection of the target RNA.

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