Annular RNA label-free detection biosensor and circRNA detection method thereof

Abstract

The invention discloses a circular RNA label-free detection biosensor and a method for detecting circRNA by the circular RNA label-free detection biosensor, the circular RNA label-free detection biosensor comprises the following components: a three-way connection TWJ probe which is used for specifically recognizing a back splicing connection BSJ sequence of circRNA, a three-way connection TWJ probe which is used for specifically recognizing a back splicing connection BSJ sequence of circRNA, and a three-way connection TWJ probe which is used for specifically recognizing a back splicing connection BSJ sequence of circRNA; the TWJ-arm probe is designed to have an unmatched basic group at the 3'terminal of the linear RNA so as to inhibit the interference of the linear RNA; the EXPAR template is used for triggering an index amplification reaction; and the transcription amplification system comprises a t7-aptamer probe and is used for generating a fluorescence signal. The circular RNA label-free detection biosensor disclosed by the invention has the beneficial effects of high sensitivity, high specificity, label-free detection, real-time detection and the like.

Description

Technical Field

[0001] The present invention relates to a label-free detection biosensor for circular RNA and a method for detecting circRNA, which is used to achieve highly sensitive and highly specific detection of circular RNA. Background Art

[0002] Circular RNAs (circRNAs) are a class of non-coding RNAs with covalently closed-loop structures and are considered potential biomarkers due to their high stability and resistance to degradation. However, due to their low abundance and sequence similarity to linear RNAs, the detection of circRNAs faces challenges. Existing detection methods such as RNA sequencing and RT-qPCR require expensive equipment and complex operation steps and are easily interfered by linear RNAs. Summary of the Invention

[0003] In view of this, one of the objectives of the present invention is to provide a label-free detection biosensor for circular RNA to achieve highly sensitive and highly specific detection of circRNAs.

[0004] The present invention solves the above technical problems through the following technical means:

[0005] A label-free detection biosensor for circular RNA of the present invention comprises the following components:

[0006] A three-way junction (TWJ) probe for specifically recognizing the back-spliced junction (BSJ) sequence of circRNA; a TWJ-arm probe designed with mismatched bases at the 3' end of linear RNA to inhibit the interference of linear RNA; an EXPAR template for triggering an exponential amplification reaction; a transcription amplification system including a t7-aptamer probe for generating a fluorescence signal.

[0007] Further, the TWJ probe includes: a target binding region for recognizing the BSJ sequence of circRNA; a short complementary sequence of the TWJ-priming probe; a recognition site for the nicking endonuclease Nt.AlwI; a track for sequence extension.

[0008] Further, the TWJ-arm probe includes: a target binding region for recognizing the BSJ sequence of circRNA; a short complementary sequence of the TWJ-priming probe; a recognition site for the nicking endonuclease Nt.AlwI.

[0009] Further, the transcription amplification system further includes: a t7 promoter sequence for initiating the transcription of a fluorescent RNA aptamer; a complementary sequence capable of binding to the x strand.

[0010] Another objective of the present invention is to provide a method for detecting circRNA by using the above biosensor.

[0011] The method for detecting circRNA of the present invention comprises the following steps: forming a TWJ structure; triggering an EXPAR reaction; generating a fluorescence signal through a transcription amplification system.

[0012] Furthermore, the method for forming the TWJ structure is as follows: adding a TWJ probe and a TWJ-arm probe to a reaction mixture, and hybridizing them with the BSJ sequence of circRNA in a sample to form a stable TWJ structure.

[0013] Furthermore, the method for triggering the EXPAR reaction is as follows: adding an EXPAR template, Klenow fragment, and Nt.AlwI enzyme to the reaction mixture, and releasing the trigger x through cyclic extension, cleavage, and strand displacement processes.

[0014] Furthermore, the method for transcription amplification is as follows: adding a t7-aptamer probe and t7 RNA polymerase to trigger a transcription amplification reaction to generate a fluorescent RNA aptamer. The beneficial effects of the present invention:

[0015] The label-free detection biosensor for circular RNA of the present invention has the following beneficial effects:

[0016] High sensitivity: By combining an exponential amplification reaction (EXPAR) and a transcription amplification system, the biosensor can achieve ultrasensitive circRNA detection with a detection limit as low as 239.605 fM.

[0017] High specificity: By designing a TWJ probe and a TWJ-arm probe and combining a base mismatch strategy, circRNA and homologous linear RNA can be effectively distinguished, reducing non-specific amplification and improving the specificity of detection.

[0018] Label-free detection: Using a fluorescent RNA aptamer as signal output, the use of traditional labeled probes is avoided, simplifying the experimental procedure, reducing costs, and improving the signal-to-noise ratio.

[0019] Real-time detection: The biosensor can perform real-time detection in a single tube, suitable for rapid and simple analysis, and is suitable for point-of-care diagnosis and on-site applications.

[0020] Simplified operation: By eliminating complex RNA processing steps such as RNase treatment, the experimental operation is simplified, reducing the experimental time and complexity.

[0021] Wide applicability: This technology is applicable to the detection of complex biological samples such as human serum and cancer cell lines, demonstrating its wide application potential in biomedical research and clinical precision medicine.

[0022] High accuracy and repeatability: The application in actual samples verifies the high accuracy and repeatability of this method, which is suitable for quantitative analysis and clinical applications.

[0023] These beneficial effects endow this biosensor with significant advantages and application prospects in the field of circRNA detection. Brief Description of the Drawings

[0024] The present invention will be further described below in conjunction with the drawings and embodiments.

[0025] Figure 1 : Schematic diagram of the T-ELAT strategy;

[0026] Figure 2 : Evaluation of fluorescence signal output based on transcription;

[0027] Figure 3 : Verification of the T-ELAT biosensor;

[0028] Figure 4 : Optimization of experimental conditions;

[0029] Figure 5 : Specificity of the T-ELAT system for circRNA detection;

[0030] Figure 6 : Analytical performance of the T-ELAT system for circmto1 detection. Detailed Embodiments

[0031] The present invention will be described in detail below in conjunction with the drawings:

[0032] 1. Construction of the biosensor T-ELAT

[0033] As Figures 1 to 6 shown, the biosensor T-ELAT of the present invention is based on the three-way junction TWJ strategy of base mismatch engineering, combined with the exponential amplification reaction EXPAR and the transcription amplification system, for label-free and real-time detection of circRNA. The biosensor includes four key probes: the TWJ-Arm probe, the TWJ-Primer probe, the t probe, and the t7-aptamer probe.

[0034] TWJ-Arm probe: Designed to recognize the back-spliced junction BSJ sequence of circRNA and has mismatched bases at the 3' end of linear RNA to inhibit the interference of linear RNA.

[0035] TWJ-Primer probe: Complementary to the TWJ-Arm probe and used to form the TWJ structure.

[0036] t probe: Serves as a template for EXPAR to trigger the exponential amplification reaction.

[0037] T7 - aptamer probe: A transcription template containing a T7 promoter sequence and a fluorescent RNA aptamer for generating a fluorescent signal.

[0038] This biosensor converts the TWJ formation event into oligonucleotide barcodes, which then trigger EXPAR to generate additional barcodes. These barcodes simultaneously activate the transcription amplification system to produce luminescent RNA aptamers that emit fluorescent signals. In this biosensor, the triggered EXPAR serves as a secondary amplification mechanism, while the transcription amplification system based on luminescent nucleic acid aptamers acts as both a signal amplifier and a fluorescent indicator. By integrating these two amplification strategies, this biosensor significantly improves the specificity and sensitivity of circRNA detection, providing a simple, label - free, and efficient method for one - pot real - time analysis.

[0039] 2. Detection steps

[0040] Sample preparation: Provide a sample containing the circRNA to be detected, such as cell lysate or serum. The sample can be prepared by conventional methods, such as using a total RNA extraction kit. Specifically, oligonucleotides are synthesized by Sangon Biotech, Shanghai, China, and purified by HPLC. The reagent of magnesium chloride hexahydrate is obtained from Sangon Biotech. DNA ladder, loading buffer, and GelRed dye are from TaKaRa Biotech. All chemicals are of reagent grade, and solutions are prepared with sterilized ddH2O.

[0041] Form the TWJ structure: Add the TWJ - Arm probe and the TWJ - Primer probe to the reaction mixture to hybridize with the BSJ sequence of circRNA in the sample, forming a stable TWJ structure.

[0042] Trigger the EXPAR reaction: Add the t - probe, Klenow fragment (3’ - 5‘ exo -), and Nt.AlwI enzyme to the reaction mixture. Through the processes of cycle extension, cleavage, and strand displacement, the trigger x is released.

[0043] Transcription amplification: Add the t7 - aptamer probe and t7 RNA polymerase to trigger the transcription amplification reaction to produce fluorescent RNA aptamers. The fluorescent RNA aptamers generate fluorescent signals after binding to fluorescent dyes.

[0044] Fluorescent signal detection: Use a real - time fluorescence detection system to monitor the change of fluorescent signals and record the curve of fluorescence intensity over time.

[0045] Data analysis: Analyze the fluorescent signals to determine the concentration of circRNA in the sample. By comparing samples with different concentrations of circRNA, evaluate the sensitivity and detection limit of the detection method. As Figure 2As shown Figure 2 is the evaluation based on the fluorescence signal output of transcription. (a) shows the real-time fluorescence responses of T7 promoter probes at different concentrations (100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 500 pM). (b) shows the linear regression analysis of the T7 promoter probe concentration and fluorescence signal. (c) shows the real-time fluorescence responses of trigger X at different concentrations (100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 500 pM). (d) shows the linear regression analysis of the trigger X concentration and fluorescence signal. (e) shows the real-time fluorescence response of alkaline SDA and T-ELAT reactions. (f) shows the comparative analysis of the fluorescence signals between basic SDA and T-ELAT reactions. Error bars represent the mean ± standard deviation (SD) of three independent experiments.

[0046] 3. Optimization of experimental conditions

[0047] To achieve the best detection performance, key reaction parameters such as the complementary sequence length, probe concentration, and reaction buffer composition were systematically optimized. For example:

[0048] Complementary sequence length: The optimal complementary base pairing length between the TWJ-Arm probe and the TWJ-Primer probe was determined to be 7 bases through experiments.

[0049] Probe concentration: The concentrations of the TWJ probe, T7-aptamer probe, and t probe were optimized and determined to be 10 nM, 50 nM, and 2.5 nM, respectively.

[0050] Reaction buffer composition: The combination of 0.25X transcription buffer and 0.75X rCutSmart buffer was selected to obtain the highest fluorescence signal.

[0051] 4. Specificity verification

[0052] The specificity of the biosensor was verified through experiments to ensure that it can effectively distinguish circRNA from homologous linear RNA. The experimental results show that even in a complex biological system, the sensor can maintain high specificity and low background signal.

[0053] 5. Practical applications

[0054] To evaluate the practical applicability of the biosensor, a recovery test was conducted using simulated clinical samples. The results show that the system has high accuracy and repeatability in quantifying circRNA in human serum. In addition, circRNA was successfully detected in total RNA extracted from different cancer cell lines, verifying the application potential of this method in different cell lines.

[0055] Through these specific embodiments, the biosensor of the present invention demonstrates its high efficiency and practicality in circRNA detection.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A circular RNA label-free detection biosensor, characterized in that: Includes the following components: include: Base mismatch engineered three-way junction (TWJ) probe set, including TWJ-Arm probe and TWJ-Primer probe; EXPAR amplification template, used to trigger the exponential amplification reaction; Transcription amplification system, including T7 promoter probe and fluorescent RNA aptamer; The 3' end of the TWJ-Arm probe contains a specific mismatch base pair for linear RNA.

2. The biosensor according to claim 1, characterized in that: The TWJ-Arm probe contains a target binding domain, a TWJ-Primer complementary domain and a nuclease recognition site; the TWJ-Primer probe contains a target binding domain and a TWJ-Arm complementary domain; the EXPAR template is an X'-N'-X' structure; and the T7 promoter probe contains a T7 promoter sequence and an X-chain complementary domain.

3. The biosensor according to claim 1, characterized in that: The 3' end of the TWJ-Arm probe is provided with 1-3 mismatched bases, and the mismatched positions are located at the end of the linear RNA binding region; the mismatched combination is selected from one or more of A / C, G / T, and T / A.

4. The biosensor according to claim 1, characterized in that: The EXPAR amplification reaction system comprises Klenow fragment, namely 3'-5' exonuclease activity and Nt.AlwI endonuclease; the transcription amplification system comprises T7 RNA polymerase and fluorescent RNA aptamer Mango.

5. A circular RNA detection method, characterized in that: The following steps are involved: a) mixing the biosensor according to claim 1 with a sample to be tested and incubating at 37° C.; b) monitoring the fluorescence signal generated by EXPAR amplification by real-time fluorescence; c) Quantify the concentration of circRNA based on the fluorescence signal intensity.

6. The method according to claim 5, characterized in that: The detection limit is 239.6 fM; The linear detection range is 1pM to 2.5nM; CircRNAs can be distinguished from their cognate linear RNAs even when the concentration of the linear RNA is up to 10 times that of the circRNA.

7. Use of the biosensor according to claim 1 in preparing a cancer diagnosis kit, characterized in that: Detection of circMTO1; suitable for complex biological samples such as serum and cell lysate.

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