Novel multiple microRNA detection method based on RNA fluorescent aptamer and exponential amplification reaction

Abstract

The invention belongs to the technical field of biological detection, and discloses a novel multiple microRNA detection method based on an RNA fluorescent aptamer and an exponential amplification reaction. According to the method, EXPAR efficient amplification and RNA fluorescent aptamer specific signal transduction are integrated, and an FLAP-EXPAR detection system is constructed. The target microRNA triggers an EXPAR reaction, a short DNA trigger chain is generated exponentially, the RNA fluorescent aptamer is further driven to transcribe, and a transcription product is combined with a specific fluorescent group to generate a target dependent fluorescence signal. By designing an exclusive combination of an amplification template, a transcription template, a fluorescent aptamer and a fluorophore, multiple microRNAs can be synchronously detected by a single tube. The platform is high in sensitivity, strong in specificity and simple and convenient to operate, the miR-21 detection limit is as low as 0.12 pM, the linear range is 1 pM-50 nM, the stability in complex matrixes such as serum and cell lysis buffer is good, the platform can be widely applied to clinical scenes such as cancer early diagnosis and prognosis evaluation, an accurate detection tool is provided for tumor molecular typing, and the application prospect is wide. The obvious clinical transformation value and the market application prospect are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a multiplex microRNA detection method based on RNA fluorescent aptamers and exponential amplification reaction (EXPAR), which is particularly suitable for the highly sensitive and specific detection of breast cancer-related microRNAs. Background Technology

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded RNA molecules with a length of approximately 18-23 nucleotides. They are widely involved in physiological processes such as cell proliferation, differentiation, and apoptosis. Abnormal expression of microRNAs is closely related to the occurrence and development of diseases such as cancer, and they are important biomarkers for early cancer diagnosis and prognostic assessment.

[0003] However, miRNAs are characterized by small molecular weight, high sequence homology, low abundance, and poor stability, posing a significant challenge to sensitive and specific detection. Traditional detection methods, such as Northern blotting, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and gene chip technology, suffer from drawbacks such as cumbersome operation, high instrument requirements, insufficient specificity, and background signal interference.

[0004] Isothermal amplification (APA) technology has become an important technique for miRNA detection due to its advantages such as high amplification efficiency, mild reaction conditions, and simple operation. Exponential amplification reaction (EXPAR) is a typical isothermal amplification technique that achieves exponential amplification of target nucleic acids through nicking enzyme cleavage and polymerase chain displacement reaction. However, traditional EXPAR detection systems use non-specific fluorescent dyes such as SYBR Green I, which suffer from high background fluorescence and low signal-to-noise ratio, seriously affecting detection sensitivity and accuracy.

[0005] RNA fluorophores are short RNA sequences with specific three-dimensional structures that can bind to specific small molecule fluorescent groups and activate fluorescence. Their fluorescence signals are target-dependent and can effectively reduce background noise. Combining RNA fluorophores with EXPAR technology is expected to overcome the inherent defects of traditional EXPAR detection systems and achieve highly sensitive and specific detection of miRNAs. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a multiplex microRNA detection method based on RNA fluorescent aptamers and EXPAR. Figure 1This method combines the high-efficiency amplification capability of EXPAR with the specific signal transduction capability of RNA fluorescent aptamers to construct a FLAP-EXPAR detection system, which can realize the simultaneous detection of multiple miRNAs in a single tube and has the characteristics of high sensitivity, strong specificity and simple operation.

[0007] 1. Components of the detection method The detection method of this invention comprises four core components: amplification template, transcription template, enzyme mixture, and fluorescent group. The functions and structures of each component are as follows: Amplification template: The sequence structure is X′–N–X′, where X′ is a recognition sequence complementary to the target miRNA and can specifically bind to the target miRNA; N is the recognition site of the Nt.BsmAI nickase, which is used to initiate the EXPAR amplification reaction.

[0008] Transcription template: The sequence structure is X′–T–B, where X′ is complementary to the EXPAR amplification product; T is the promoter sequence recognized by T7 RNA polymerase; B is the RNA fluorescent aptamer coding sequence, used to transcribe and generate specific RNA fluorescent aptamers.

[0009] Enzyme mixture: Contains Klenow fragment polymerase, Nt.BsmAI nickase, T7 RNA polymerase, and ribonuclease inhibitor. Klenow fragment polymerase is used for DNA strand elongation; Nt.BsmAI nickase is used to cleave the DNA double strand to generate new amplification primers; T7 RNA polymerase drives RNA fluorescent aptamer transcription; ribonuclease inhibitor prevents RNA fluorescent aptamer degradation.

[0010] Fluorescent groups: Small molecules that specifically bind to RNA fluorophores and produce fluorescence only after binding, effectively reducing background noise. DFHBI-1T, malachite green, and HBC620 are preferred, corresponding to the three RNA fluorophores Broccoli, MGA, and Pepper, respectively.

[0011] 2. Steps of the detection method The detection method of this invention is a one-pot reaction, eliminating the need for cumbersome intermediate processing steps, specifically including: Reaction system preparation: Prepare the reaction mixture and enzyme mixture separately on ice. The reaction mixture contains amplification template, target sample, dNTPs, DTT, MgSO4, rCutSmart buffer, and RNase-free water; the enzyme mixture contains transcription template, NTPs, fluorescent groups, various enzyme preparations, and RNase-free water.

[0012] One-pot reaction: The two mixtures are rapidly combined and incubated at 37°C for 100 min. The target miRNA first triggers the EXPAR amplification reaction, exponentially generating a short DNA trigger strand; the trigger strand binds to the transcription template, initiating a T7 RNA polymerase-mediated transcription reaction, generating an RNA aptamer; the RNA aptamer binds to a fluorescent group, producing a specific fluorescent signal.

[0013] Fluorescence signal detection: A real-time quantitative PCR instrument was used to collect fluorescence signals in the detection channels of the corresponding fluorescent groups. Qualitative analysis was performed based on the presence or absence of fluorescence signals, and quantitative analysis was performed based on the linear relationship between fluorescence signal intensity and miRNA concentration.

[0014] 2. Feasibility verification testing We first selected miR-21 as the model target to evaluate the feasibility of the FLEXPAR system. A corresponding EXPAR template was designed to specifically recognize miR-21 and initiate the amplification reaction. As shown in Figure 2, the target miRNA hybridizes with the amplification template, triggering a polymerase-mediated chain elongation reaction and a nicking enzyme-assisted cleavage reaction, generating a short DNA strand of similar length to the miRNA. The EXPAR process was first verified using non-denaturing polyacrylamide gel electrophoresis (PAGE). Figure 3 As shown in Figure A, in the absence of the target miRNA, only a single band corresponding to the amplification template appeared on the gel (lane 3); however, when miR-21 was present, two additional bands appeared on the gel (lane 4), corresponding to the single-stranded product and a slightly longer double-stranded complex, respectively, indicating that the target-triggered amplification reaction proceeded successfully. Real-time fluorescence monitoring further verified the initiation of EXPAR (Figure 3B). Significant fluorescence enhancement was only observed in the presence of miR-21, while the signal change in the negative control was negligible, indicating minimal background amplification.

[0015] To verify the feasibility of transcription-based signal transduction, a simulated EXPAR product was introduced into the transcription reaction system. Figure 4As shown in Figure 5A, no RNA product was detected in the absence of T7 RNA polymerase; however, after the addition of the enzyme, a distinct RNA band appeared, and this band remained after DNase I digestion, confirming the successful in vitro transcription reaction. The presence of multiple RNA bands is due to premature termination of transcription caused by secondary structures within the template. This study also compared the transcription efficiency of the traditional T7 promoter system with the FLEXPAR-triggered promoter generation strategy. Although the traditional system produced higher absolute fluorescence intensity, the FLEXPAR strategy significantly reduced nonspecific background, thereby improving signal fidelity. Figure 5 B).

[0016] Finally, the feasibility of the single-tube FLEXPAR system was verified by combining EXPAR amplification and transcription reactions in a single tube. PAGE analysis and real-time fluorescence monitoring were also performed. Figure 6 This confirms that EXPAR and the transcriptional response can be successfully activated sequentially, indicating that the integrated single-tube detection method has good compatibility and specificity.

[0017] To differentiate the kinetics of EXPAR amplification and transcription reactions in a single-tube system, this study used the non-specific nucleic acid dye SYBR and the Broccoli RNA aptamer-specific dye DFHBI-1T for real-time monitoring of the reactions (Figure 6B). The results showed that SYBR fluorescence, reflecting total nucleic acid accumulation, rapidly increased in the early stages of the reaction (within 5 minutes), reaching a plateau around 15 minutes, and its final signal intensity was significantly higher than that of DFHBI-1T. Conversely, DFHBI-1T fluorescence, representing the generation of target Broccoli RNA, increased more slowly, and its signal intensity remained lower than that of SYBR throughout the reaction. This difference arises because SYBR can bind to all double-stranded nucleic acids in the system, including DNA intermediates generated during EXPAR amplification and the reaction template itself. Therefore, SYBR fluorescence directly reflects the efficient DNA amplification capability of EXPAR. When the target miRNA activates EXPAR, a large amount of DNA product is rapidly generated through KF polymerase-mediated strand displacement and cyclic cleavage by nucleases. DFHBI-1T binds only to Broccoli RNA produced during the transcription phase, and its fluorescent signal generation depends on a series of cascade reactions: the EXPAR product binds to the transcription template, which then elongates to form the T7 promoter, followed by T7 RNA polymerase (RNAP) initiating the transcription reaction. This transcription process is the rate-limiting step in the entire system, requiring multiple coupled enzymatic reactions to proceed in concert. Therefore, compared to SYBR, the DFHBI-1T signal rises more slowly and reaches a lower intensity. However, the detectable DFHBI-1T signal indicates that downstream transcription can only proceed effectively after EXPAR activation, highlighting the system's specific response to the target product.

[0018] 3. Optimization parameters of the detection method To achieve the best detection results, the key parameters of the reaction system were optimized, and the optimal parameters were determined as follows: A MgSO4 concentration of 15 mM ensures optimal activity of the three enzymes. Figure 7 A); Enzyme concentration: Nt.BsmAI nickase 0.2 U / μL ( Figure 7 B), Klenow fragment polymerase 0.05 U / μL ( Figure 7 C), T7 RNA polymerase 1.5 U / μL ( Figure 7 D), to avoid nonspecific amplification; Template concentration: 5 nM of amplified template ( Figure 8 A) Transcription template 20 nM ( Figure 8 B, 8C), balancing signal strength and signal-to-noise ratio.

[0019] 4. Sensitivity and specificity assessment of FLEXPAR We systematically evaluated the analytical performance of the FLEXPAR system for miR-21 in terms of both sensitivity and specificity. As shown in Figure 9A, the real-time fluorescence curves exhibited a clear concentration dependence. With the miR-21 concentration increasing from 1 pM to 50 nM, both the fluorescence rise rate and the final signal intensity gradually increased, while the negative control (NC) maintained a baseline signal throughout the reaction. This result indicates that the initiation efficiency of the EXPAR-transcriptional cascade reaction is directly determined by the concentration of the target miRNA.

[0020] At higher miR-21 concentrations, successive EXPAR cycles generate more trigger strands, which subsequently promote the transcription of Broccoli RNA aptamers. When these RNA aptamers bind to DFHBI-1T, an enhanced fluorescence signal is produced. Within the concentration range of 1 pM to 50 nM, a good linear relationship exists between the logarithm of miR-21 concentration and the logarithm of the fluorescence response, with a correlation coefficient (R²) of 0.9993. Figure 9 B). The limit of detection (LOD) of this method is approximately 0.12 pM. These results demonstrate that the FLEXPAR platform possesses excellent quantification capabilities and a wide dynamic detection range.

[0021] The system is highly sensitive because the efficient DNA amplification mediated by EXPAR and the downstream RNA aptamer-based signal transduction work synergistically to achieve effective signal amplification of trace amounts of target miRNA.

[0022] To further investigate the specificity of the FLEXPAR system, this study used 100 pM concentrations of non-target miRNAs and mismatched single-, double-, and triple-base sequences of miR-21 as interfering agents for detection (Figure 10). In all these control experiments, only weak fluorescence responses were observed. Conversely, perfectly matched miR-21 elicited significant fluorescence enhancement. This high selectivity stems from the EXPAR sequence-dependent initiation mechanism, which requires the target miRNA to be perfectly complementary to the amplification template to trigger strand elongation and cyclic cleavage reactions. Mismatched miRNAs or non-target miRNAs cannot effectively initiate the EXPAR process, thereby inhibiting downstream transcriptional reactions and fluorescence generation. In summary, these results confirm that the FLEXPAR system can achieve highly sensitive quantitative detection and reliable sequence-specific differentiation of miR-21.

[0023] 5. Feasibility verification of multiple detection methods Given the superior analytical performance of the FLEXPAR platform, we further explored its ability to simultaneously detect multiple miRNA targets. Three breast cancer-related miRNAs (miR-21, miR-155, and miR-1246) were selected as representative targets, and they were simultaneously detected in a single reaction system. This group of targets includes clinically relevant biomarkers associated with tumor proliferation, metastasis, and immune regulation, providing a suitable model for evaluating the performance of multiplex detection.

[0024] like Figure 11 As shown, the simultaneous detection of multiple miRNAs was achieved by designing target-specific EXPAR amplification templates and corresponding transcription templates for each miRNA. Each transcription template encodes a unique RNA aptamer that selectively binds to a specific fluorophore. Specifically, the Broccoli-DFHBI-1T, MGA-MG, and Pepper-HBC620 aptamer-fluorophore pairs were assigned to miR-21, miR-155, and miR-1246, respectively. Fluorescence signals were collected using the SYBR, Cy5, and Texas Red channels of a Bio-Rad real-time quantitative PCR instrument, which are matched to the excitation and emission characteristics of the corresponding fluorophore-aptamer systems.

[0025] Figure 12Real-time fluorescence curves showed significant target-dependent signal responses in all detection channels. In the SYBR channel, significant fluorescence enhancement was observed only in the miR-21 group; similarly, significant fluorescence enhancement was observed in the miR-155 group in the Cy5 channel, and in the Texas Red channel, significant fluorescence enhancement was observed in the miR-1246 group, while the blank control group and non-target samples showed only extremely low background signals. This channel-specific fluorescence output stems from the strict complementarity between each miRNA and its corresponding amplification template. This complementarity ensures the selective initiation of the EXPAR-transcriptional cascade and the generation of the corresponding RNA aptamers.

[0026] Compared with the prior art, the present invention has the following beneficial effects: High sensitivity: Combining the advantages of EXPAR exponential amplification and low background signal of RNA fluorescent aptamers, the detection limit for miR-21 is as low as 0.12 pM, with a linear range of 1 pM-50 nM, enabling the detection of low-abundance miRNAs in biological samples.

[0027] High specificity: The specific recognition of target miRNAs by amplifying template and the specific binding of RNA fluorescent aptamers to fluorescent groups ensure detection specificity, with no obvious cross-reactivity to single-base mismatch sequences.

[0028] Multiplex detection capability: Through a proprietary combination design, the single-reaction system can simultaneously detect multiple miRNAs with no signal crosstalk between channels, significantly improving detection efficiency.

[0029] Simple to operate: One-pot reaction mode, no need for complicated sample processing, constant temperature reaction at 37℃, no need for expensive and precision instruments, easy to promote in clinical practice.

[0030] Strong anti-interference ability: The recovery rate in 10% human serum samples is 96.60%-99.22%, and the relative standard deviation is less than 9%, which can be effectively applied to the detection of complex biological matrices. Attached Figure Description

[0031] Figure 1 is a schematic diagram of FLAP-EXPAR design and activation.

[0032] Figure 2 is a schematic diagram of simple EXPAR.

[0033] Figure 3 shows the feasibility analysis of EXPAR alone. (A) PAGE gel analysis diagram. (B) Real-time fluorescence curve.

[0034] Figure 4 is a schematic diagram of in vitro transcription.

[0035] Figure 5 shows the feasibility analysis of in vitro transcription. (A) PAGE gel analysis. (B) Real-time fluorescence curves of the two transcription modes.

[0036] Figure 6 shows the feasibility analysis of the FLAP-EXPAR one-pot method. (A) PAGE gel analysis diagram. (B) Real-time fluorescence curves using SYBR and DFHBI-1T as fluorescent dyes, respectively.

[0037] Figure 7 This is the result of the FLAP-EXPAR reaction condition optimization 1. (A) Fluorescence response to different concentrations of magnesium ions. (B) Fluorescence response to different concentrations of nicking enzyme. (C) Fluorescence response to different concentrations of polymerase. (D) Fluorescence response to different concentrations of T7 RNAP.

[0038] Figure 8 This is the result of FLAP-EXPAR reaction condition optimization 2. (A) Fluorescence response to different concentrations of amplified template. (B) Fluorescence response to different concentrations of transcribed template. (C) Changes in signal-to-noise ratio and positive signal value with transcribed template concentration (0-90 nM).

[0039] Figure 9 This is a sensitivity analysis of the FLAP-EXPAR system. (A) Real-time fluorescence curves of the response to different concentrations of the target miR-21. (B) Linearity results for different concentrations of the target miR-21.

[0040] Figure 10 This is the response result of the FLAP-EXPAR system to different miRNA targets.

[0041] Figure 11 This is a schematic diagram of the FLAP-EXPAR system's multiple detection.

[0042] Figure 12 These are the real-time fluorescence curves of FLAP-EXPAR for three miRNAs in three detection channels. (A) SYBR channel. (B) Texas Red channel. (C) Cy5 channel. Detailed Implementation

[0043] (1) Detection of single miRNAs:

[0044] ① Detection system configuration

[0045] Reaction mixture (10 μL): 5 nM amplification template, 1 μL target sample, 1 mM dNTPs, 1 mM DTT, 15 mM MgSO4, 1× rCutSmart buffer, and RNase-free water to make up the volume;

[0046] Enzyme mixture (10 μL): 20 nM transcription template, 0.5 mM NTP, 25 μM DFHBI-1T, 0.05 U / μL Klenow fragment polymerase, 0.2 U / μL Nt.BsmAI nickase, 1.5 U / μL T7 RNA polymerase, 1 U / μL ribonuclease inhibitor, with RNase-free water added to make up the volume.

[0047] ② Reaction and detection: The two solutions were mixed and incubated at 37℃ for 100 min. The signal was acquired using the SYBR channel of a real-time quantitative PCR instrument. The results showed that the concentration of miR-21 had a good linear relationship with fluorescence intensity in the range of 1 pM-50 nM (R²=0.9993), and the detection limit was 0.12 pM.

[0048] (2) Multiplex miRNA detection

[0049] ① Detection system configuration

[0050] Reaction mixture (10 μL): 5 nM miR-21 amplification template, 5 nM miR-155 amplification template, 5 nM miR-1246 amplification template, 1 μL target sample, 1 mM dNTPs, 1 mM DTT, 15 mM MgSO4, 1× rCutSmart buffer, RNase-free water to make up the volume;

[0051] Enzyme mixture (10 μL): 20 nM miR-21 transcription template (encoding Broccoli), 20 nM miR-155 transcription template (encoding MGA), 20 nM miR-1246 transcription template (encoding Pepper), 0.5 mM NTP, 25 μM DFHBI-1T, 25 μM malachite green, 25 μM HBC620, 0.05 U / μL Klenow fragment polymerase, 0.2 U / μL Nt.BsmAI nickase, 1.5 U / μL T7 RNA polymerase, 1 U / μL ribonuclease inhibitor, RNase-free water to make up the volume.

[0052] ②Reaction and detection: Mix the two solutions and incubate at 37℃ for 100 min. Collect signals in the SYBR (miR-21), Cy5 (miR-155), and Texas Red (miR-1246) channels respectively. The detection signals of the three miRNAs do not interfere with each other and can all achieve sensitive quantification.

[0053] (3) Detection of miRNA in serum samples ① Sample processing: Human serum was diluted 10 times with RNase-free water to prepare spiked samples containing different concentrations of miR-21; ② Preparation of the detection system: Refer to the system in Example 1, and replace the target sample with a diluted serum sample; ③ Reaction and detection: Following the steps in Example 1, the recovery rate of miR-21 in serum samples was 96.60%-99.22%, with a relative standard deviation of less than 9%, indicating that the detection system has strong anti-interference ability.

Claims

1. A novel method for detecting multiplex microRNAs based on RNA fluorescent aptamers and exponential amplification reactions, characterized in that, Includes amplification template, transcription template, enzyme mixture, and fluorescent dye: (1) The amplification template contains a target microRNA recognition region and a nickase recognition site, and its sequence structure is X′–N–X′, where X′ is a sequence complementary to the target microRNA and N is the recognition site of the Nt.BsmAI nickase; (2) The transcription template contains a complementary recognition sequence, a T7 promoter region and an RNA fluorescent aptamer coding sequence, and its sequence structure is X′–T–B, where T is the T7 promoter sequence and B is the RNA fluorescent aptamer coding sequence; (3) The enzyme mixture contains Klenow fragment polymerase (3'→5' exo-), Nt.BsmAI nickase, T7 RNA polymerase and ribonuclease inhibitor; (4) The fluorescent group is a small molecule that specifically binds to RNA fluorescent aptamers, including one or more of DFHBI-1T, malachite green, and HBC620.

2. The detection method according to claim 1, characterized in that, The RNA fluorescent aptamers include one or more of Broccoli, MGA, and Pepper, with different RNA fluorescent aptamers corresponding to different target microRNAs and specific fluorescent groups.

3. A method for detecting multiplex microRNAs based on the detection method of claim 1, characterized in that, Includes the following steps: (1) Preparation of reaction system: Prepare reaction mixture and enzyme mixture on ice. The reaction mixture contains amplification template, target sample, dNTPs, DTT, MgSO4, rCutSmart buffer and RNase-free water; the enzyme mixture contains transcription template, NTPs, fluorescent group, Klenow fragment polymerase, Nt.BsmAI nickase, T7 RNA polymerase, ribonuclease inhibitor and RNase-free water. (2) One-pot reaction: The reaction mixture and enzyme mixture are rapidly mixed and incubated at 37°C for 90-120 min; (3) Fluorescence signal detection: A real-time fluorescence quantitative PCR instrument is used to collect fluorescence signals in the detection channels of the corresponding fluorescent groups. The qualitative and quantitative analysis of the target microRNA is realized based on the changes in fluorescence signals.

4. The detection method according to claim 3, characterized in that, The final concentration parameters of the reaction system described in step (1) are as follows: amplification template concentration 5 nM, transcription template concentration 20 nM, dNTPs concentration 1 mM, MgSO4 concentration 15 mM, Klenow fragment polymerase concentration 0.05 U / μL, Nt.BsmAI nickase concentration 0.2 U / μL, T7 RNA polymerase concentration 1.5 U / μL, ribonuclease inhibitor concentration 1 U / μL, and fluorescent group concentration 25 μM.

5. The detection method according to claim 3, characterized in that, The incubation time in step (2) is preferably 100 min, which ensures that the EXPAR amplification reaction and the downstream transcription reaction can proceed fully.

6. The detection method according to claim 3, characterized in that, The multiplex microRNA detection described in step (3) is achieved by assigning specific combinations of "amplification template-transcription template-RNA fluorescent aptamer-fluorescent group" to different target microRNAs. Different combinations correspond to different fluorescence detection channels, and there is no obvious signal crosstalk between channels.

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