Modularized fluorescent RNA aptamer biosensor system

Abstract

The invention discloses a modular fluorescent RNA aptamer biosensor system, and relates to the field of medicine. Comprising a probe module, an enzyme system, a dye and a reaction buffer solution, the probe module comprises a promoter probe P and a reporter probe R, the enzyme system comprises SplintR ligase and T7RNA polymerase, and the reaction buffer solution comprises a transcription buffer solution and a SplintR ligase buffer solution. The promoter probe P comprises a T7 promoter sequence and a 5 '-phosphorylated upstream recognition region, and the length of the promoter probe P is 10nt. According to the present invention, the ligase-assisted probe assembly and the T7RNA polymerase-mediated fluorescent RNA aptamer transcription are integrated, the simultaneous detection of the multiple circRNA can be achieved without the complex probe labeling, the femtomole-level sensitivity and the excellent single base mutation distinguishing ability in the complex sample are provided, and the real-time fluorescence instrument and the portable device are adapted.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a modular fluorescent RNA aptamer biosensor system. Background Art

[0002] The emergence of circular RNA (circRNA) has revolutionized our understanding of gene expression and regulation, particularly in cancer biology. Multiplexed circRNA detection panels have demonstrated higher accuracy in diagnosis and prognosis compared to single biomarkers.

[0003] Circular RNAs (circRNAs) are a class of non-coding RNAs with covalently closed circular structures, high stability, and cell-type-specific expression. They are widely considered to be important biomarkers for cancer diagnosis and treatment monitoring. Studies have shown that multiple circRNA combinations have higher diagnostic and prognostic capabilities than single markers. Current mainstream detection methods, such as qRT-PCR and chip technology, are complex and unsuitable for convenient and rapid on-site testing. Biosensors based on isothermal amplification have attracted much attention due to their simplicity, high sensitivity, and suitability for single-tube reactions. Although strategies such as HCR, CRISPR-PER, LAMP, and RCA have been developed to achieve single-target detection, multi-target detection still requires fluorescently labeled probes, limiting its practicality. In recent years, fluorescent RNA aptamers (FRAs) have emerged as label-free signal output methods in detection, such as Spinach and Mango, but label-free single-tube platforms for multi-target detection remain unavailable.

[0004] To this end, the present invention proposes a modular fluorescent RNA aptamer biosensor system. Summary of the Invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a modular fluorescent RNA aptamer biosensor system.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A modular fluorescent RNA aptamer biosensor system comprises a probe module, an enzyme system, a dye and a reaction buffer, wherein the probe module comprises a promoter probe P probe and a reporter probe R probe, the enzyme system comprises SplintR ligase and T7 RNA polymerase, and the reaction buffer comprises a transcription buffer and a SplintR ligase buffer.

[0008] Preferably, the promoter probe P probe includes a T7 promoter sequence and a 5'-phosphorylated upstream recognition region, and has a length of 10 nt.

[0009] Preferably, the reporter probe R probe comprises a 3'-downstream recognition region and a signal region encoding a fluorescent RNA aptamer.

[0010] Preferably, the 3'-downstream recognition region is 10 nt in length, and the fluorescent RNA aptamer is iSpinach or Mango.

[0011] Preferably, the ratio of the reaction buffer is: 25U transcription buffer and 50U SplintR ligase buffer.

[0012] Preferably, the transcription buffer comprises: 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine.

[0013] Preferably, the SplintR ligase buffer comprises 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, and 10 mM DTT.

[0014] Preferably, the detection principle of the modular fluorescent RNA aptamer biosensor system is:

[0015] S1: When the target circRNA is present, the P probe and the R probe are adjacent through the back-splicing site BSJ;

[0016] S2: SplintR ligase catalyzes the ligation of probes to form a complete DNA template;

[0017] S3: T7 RNA polymerase transcribes and generates fluorescent RNA aptamers, which emit fluorescent signals after binding to the dye;

[0018] S4: No template is formed and no signal is output when there is no circRNA.

[0019] The beneficial effects of the present invention are:

[0020] 1. This invention integrates ligase-assisted probe assembly with T7 RNA polymerase-mediated transcription of fluorescent RNA aptamers, enabling simultaneous detection of multiple circRNAs without the need for complex probe labeling. It exhibits femtomolar sensitivity and excellent single-base mutation discrimination in complex samples, and is compatible with real-time fluorescence instruments and portable devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a detection principle diagram of a modular fluorescent RNA aptamer biosensor system proposed in the present invention. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0023] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting", "setting" should be understood broadly, for example, it can be fixedly connected, set, or it can be detachably connected, set, or integrally connected, set. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0024] Embodiments

[0025] Oligonucleotides and reagents

[0026] All oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. and purified by high-performance liquid chromatography (HPLC). The following reagents were purchased from commercial channels: T7 RNA Polymerase (50,000 U / mL), SplintR Ligase (25,000 U / mL), rNTPs (25 mM), and dNTPs (100 mM) were all purchased from New England Biolabs (China). TO1-biotin was purchased from Applied Biological Materials (China), and DFHBI-1T was purchased from MedChemExpress (China). Mixed human serum was purchased from Ruibao Biological (Shenzhen). High-Performance qRT MasterMix (gDNA Remove) and 2x Universal SYBR qPCR Master Mix were purchased from YoungGen (Kunming). FastPure cell / tissue total RNA Isolation Kit V2 was purchased from Vazyme (Nanjing). Human cell lines T24, RT4 and PC3 were purchased from Ponsay Biotech (Wuhan).

[0027] One-tube detection process

[0028] The total volume of a single-tube reaction system was 20 μL and included: 5 nM R probe, 5 nM P probe, 1× transcription buffer (40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine), 1× SplintR ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT), 0.5 mM DFHBI-1T, 0.5 mM TO1-biotin, 1.25 mM rNTPs, 25 U of SplintR ligase, and 50 U of T7 RNA polymerase. Different concentrations of target circRNA were added according to experimental requirements. Reactions were performed at 37°C in a real-time fluorescence PCR system (e.g., Bio-Rad CFX96), and fluorescence signals were recorded every 30 seconds in the FAM and HEX channels. For endpoint detection, 10 μL of the reaction solution was added dropwise onto a circular filter paper and photographed using a digital camera under 365 nm UV light, or using a multifunctional gel imaging system (O1600MF, China) under blue or green light excitation.

[0029] Polyacrylamide gel electrophoresis (PAGE) analysis

[0030] To verify the specificity of the ligation and transcription products, 10 μL of the reaction mixture was subjected to in situ polyacrylamide gel analysis. A 12% polyacrylamide gel (acrylamide:bisacrylamide mass ratio of 29:1) was run in 1× TBE electrophoresis buffer at a constant voltage of 100 V for 60 minutes. The gel was stained with Gelstain Red nucleic acid dye for 30 minutes and then photographed using a Bio-Rad ChemiDoc imaging system.

[0031] Establishment of subcutaneous tumor model

[0032] Ten male BALB / c nude mice, aged 4–6 weeks and weighing 18–22 grams, were housed under specific pathogen-free (SPF) conditions with free access to food and water. All animal experiments were approved by the Institutional Animal Care and Ethics Committee (IACUC) of Chongqing Medical University and performed in accordance with national and institutional animal welfare standards.

[0033] Human bladder cancer cells T24 were cultured in McCoy's 5A medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S). When the cell density reached 80% confluence, the cells were resuspended in PBS to a volume of 2.5 × 10 7 cells / mL.

[0034] Five mice were randomly selected as the experimental group, and 200 μL of cell suspension (containing 5×10 6The length (L) and width (W) of the tumor were measured regularly, and the volume was calculated using the formula: V = 1 / 2 × L × W 2 When the maximum diameter of the tumor exceeded 2 cm, the mice were euthanized using CO2.

[0035] Clinical sample analysis

[0036] Serum sample testing: Synthetic circRNAs of known concentration were added to 5% pooled human serum and analyzed according to the standard single-tube assay protocol. Cell and tissue samples: Total RNA was extracted using the FastPure Cell / Tissue Total RNA Extraction Kit V2 according to the manufacturer's instructions. Cell line samples included human cancer cells T24, RT4, and PC3; mouse samples included xenograft tumor tissue derived from subcutaneous transplantation of T24 cells (experimental group) and major organs (heart, spleen, lung, and kidney) from healthy mice as controls.

[0037] principle

[0038] The FRA-MOML system consists of two probes: a Promoter probe (P) and a Reporter probe (R). The P probe contains a T7 promoter and a 5' phosphorylated upstream recognition region; the R probe contains a 3' downstream recognition region and a signal region encoding a fluorescent aptamer.

[0039] In the presence of the target circRNA, the two probes juxtapose at the BSJ site and are ligated by ligase to form a complete DNA template. T7 RNA polymerase then transcribes the aptamer to generate a fluorescent RNA aptamer, which then emits light upon binding to its specific dye. In the absence of the target, no template is formed, resulting in no signal. By varying the aptamer sequence in the R probe, multiple luminescent signals can be distinguished and read out in multiple channels, making it suitable for real-time PCR instruments, gel imaging systems, or portable devices such as mobile phones.

[0040] Feasibility verification of FRA-MOML biosensor

[0041] To verify the feasibility of fluorescent RNA aptamers in ligase-mediated transcription amplification, the present invention set up six groups of experiments: circRNA group, linear RNA group, no P probe group, no R probe group, no ligase group, and no target group. The results showed that significant fluorescent signals could only be detected when all key components (P probe, R probe, ligase, T7 RNA polymerase, target circRNA) were present at the same time. No significant signal enhancement was observed when any component was missing or non-target RNA was used. Digital photos under 365nm ultraviolet light excitation further confirmed that visible fluorescent signals were only observed in the circRNA group. Further verification by 12% polyacrylamide gel electrophoresis (PAGE) analysis showed that only the circRNA group showed complete transcription products, while the other groups (such as the no P probe group, linear RNA group, no R probe group, etc.) only showed partial transcription bands, indicating that no effective template was formed. To further verify the key role of T7 transcription in signal generation, the present invention designed an experimental group lacking T7 RNA polymerase. As expected, in the absence of this enzyme, no significant fluorescent signal was generated, regardless of the presence or absence of other components, highlighting the indispensable role of transcription in the entire detection mechanism.

[0042] Reaction optimization

[0043] To improve sensitivity and reduce background interference, the present invention systematically optimized reaction conditions, using 1 nM circSMARCA5 as the positive sample and no target as the negative control. The following variables were optimized individually: ligase dose: 25 U was optimal, resulting in the highest signal-to-noise ratio (S / N); T7 RNA polymerase dose: 50 U was optimal; reaction temperature: 37.1°C for optimal S / N; P and R probe concentration: 5 nM for optimal S / N; and ligation recognition region length: 10 bases for both the P and R probes. Reaction buffer conditions were also optimized. A standard 1× concentration of T7 transcription buffer and SplintR ligase buffer provided the optimal fluorescence signal. These optimizations laid a solid foundation for subsequent highly sensitive, single-tube, label-free detection.

[0044] Detection performance of FRA-MOML biosensor

[0045] In the optimized reaction system, the present invention adopts a simplified "one-tube method", that is, all reaction components are added at the same time, which simplifies the operation and reduces the risk of contamination. The fluorescence signal generated by this one-tube configuration is slightly lower than that of the two-step method, but still within an acceptable range, indicating that the system is feasible in practical applications. In order to achieve multiple detection, the present invention designed a second pair of probes P2 / R2 to identify circSLC38A1, whose transcription product is a Mango aptamer, which emits different colors of fluorescence after binding to TO1-Biotin; the original circSMARCA5 probe is recorded as P1 / R1. Target detection circSMARCA5: Concentration-dependent fluorescence enhancement from 0.5pM to 1nM; the calibration curve is y=564+50.04x(R 2 =0.9844), with a limit of detection (LOD) of 290.18 fM. circSLC38A1: detection range was 0.5 pM to 5 nM; the calibration curve was y = 54.43 + 2.62 x (R 2 =0.9621), and the LOD was 349.47 fM. This indicates that the modular sensor exhibits low femtomolar sensitivity and a wide linear range for both targets, outperforming most reported methods.

[0046] Specificity and single-base recognition ability

[0047] To evaluate specificity, the present invention tested the ability of the sensor to distinguish non-related miRNAs, other circRNAs and their linear analogs:

[0048] 1) For circSMARCA5, only circSMARCA5 itself can induce strong fluorescence, while non-related miRNAs, other circRNAs and linear RNAs all have very weak signals.

[0049] 2) circSCL38A1 also exhibited high specificity. Fluorescence imaging further confirmed that visible fluorescence was observed on the paper only in the presence of the target circRNA. In addition, the present invention constructed a series of 20nt oligonucleotide sequences containing single-base mutations:

[0050] 3) For circSMARCA5 and circSCL38A1, the closer the mutation site is to the junction site, the more obvious the fluorescence signal attenuation.

[0051] 4) The mutation resulted in a significant decrease in fluorescence, while the wild-type sequence produced the strongest signal, indicating that the platform has excellent single-base recognition capabilities.

[0052] Multiplex detection capabilities of FRA-MOML biosensors

[0053] To validate the multiplex detection capabilities of the FRA-MOML platform, the present invention simultaneously added two circRNAs: circSMARCA5 and circSCL38A1, into a single tube. Three concentration gradients were set: 1 nM, 2.5 nM, and 5 nM, respectively. Real-time fluorescence signals and visual detection images on filter paper were recorded using green or blue light excitation. At all concentration levels: if only one of the circRNA targets was added, only the fluorescence signal of the corresponding channel was significantly enhanced; if both targets were present, both channels produced significant signals; if no targets or corresponding probes were added, neither channel had a significant signal. When performing end-to-end detection on filter paper, all samples containing the targets (1 nM, 2.5 nM, 5 nM) produced visible fluorescence under green or blue light irradiation, and there was no cross-interference between the two channels: green spots indicate activation of the iSpinach aptamer after circSMARCA5 recognition; orange spots indicate activation of the Mango aptamer after circSCL38A1 recognition. The results clearly demonstrate that the modular biosensor is capable of orthogonal, non-interference detection of multiple circRNA targets in a single reaction tube, thus validating its feasibility in label-free, one-tube multiplex detection.

[0054] Practical application evaluation

[0055] To evaluate the practical application potential of the biosensor, the present invention was tested in human serum, cell lines and mouse tissue samples: serum recovery experiment

[0056] Synthetic circSMARCA5 or circSCL38A1 was spiked into 5% human serum at varying concentrations (1 nM, 0.5 nM, and 0.1 nM). The recovery rates for circSMARCA5 and circSCL38A1 ranged from 96.84% to 106.45%, and 102.81% to 107.14% respectively, with relative standard deviations (RSDs) ranging from 2.88% to 11.69%. This method demonstrates excellent accuracy and reproducibility in complex biological matrices. Cell-based assays: Total RNA was extracted from three human cancer cell lines (T24, RT4, and PC3), and the biosensor was used to measure the expression levels of circSMARCA5 and circSCL38A1. Fluorescence-based assay data were consistent with qRT-PCR results, demonstrating the suitability of this platform for quantitative analysis of circRNAs in cell samples. Tissue-based assays: T24 cells were subcutaneously inoculated in nude mice to establish a bladder cancer xenograft model. Tumor, heart, spleen, lung, and kidney tissues were collected from tumor-bearing mice and healthy controls for a single-tube assay. Results showed significantly enhanced fluorescence signals for circSMARCA5 and circSCL38A1 in tumor tissues, while signals were minimal in normal tissues and healthy controls.

[0057] The results show that the biosensor has high specificity, high sensitivity and wide applicability in animal models and clinically relevant tissues.

[0058] in conclusion

[0059] This study successfully developed a modular, label-free, single-tube, multiplexed circRNA biosensor platform. This platform combines ligase-mediated target recognition, T7 RNA polymerase-driven transcriptional amplification, and spectrally distinguishable fluorescent RNA aptamer / dye pairs (such as iSpinach / DFHBI-1T and Mango / TO-1), eliminating the need for traditional fluorescently labeled probes and enabling simultaneous quantification of multiple targets within a single tube. In bladder cancer-related circSMARCA5 and circSCL38A1 models, the platform demonstrated femtomolar sensitivity (LODs of 290 fM and 424 fM, respectively), precise identification of single-base mutations near ligation sites, and the ability to quantify multiple targets in a single tube. Furthermore, the platform utilizes a plug-and-play probe design, enabling rapid adaptation to new targets by simply replacing the probe sequence, eliminating the need for repeated reaction system optimization. Combined with the low cost of RNA aptamers and their potential for portable detection (e.g., smartphone readout), this platform is well-suited for point-of-care (POCT) applications and can be expanded to research, clinical, and primary care settings. The FRA-MOML platform not only significantly advances the development of circRNA detection technology, but also provides a promising technical means for early disease screening, diagnosis and monitoring.

[0060] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A modular fluorescent RNA aptamer biosensor system, characterized in that: The method comprises a probe module, an enzyme system, a dye and a reaction buffer, wherein the probe module comprises a promoter probe P probe and a reporter probe R probe, the enzyme system comprises SplintR ligase and T7 RNA polymerase, and the reaction buffer comprises a transcription buffer and a SplintR ligase buffer.

2. A modular fluorescent RNA aptamer biosensor system according to claim 1, characterized in that: The promoter probe P probe includes a T7 promoter sequence and a 5'-phosphorylated upstream recognition region, and has a length of 10 nt.

3. A modular fluorescent RNA aptamer biosensor system according to claim 1, characterized in that: The reporter probe R probe includes a 3'-downstream recognition region and a signal region encoding a fluorescent RNA aptamer.

4. A modular fluorescent RNA aptamer biosensor system according to claim 3, characterized in that: The 3'-downstream recognition region is 10 nt in length, and the fluorescent RNA aptamer is iSpinach or Mango.

5. A modular fluorescent RNA aptamer biosensor system according to claim 1, characterized in that: The ratio of the reaction buffer is: 25 U transcription buffer and 50 U SplintR ligase buffer.

6. A modular fluorescent RNA aptamer biosensor system according to claim 5, characterized in that: The transcription buffer comprises: 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, and 2 mM spermidine.

7. A modular fluorescent RNA aptamer biosensor system according to claim 5, characterized in that: The SplintR ligase buffer includes 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, and 10 mM DTT.

8. A modular fluorescent RNA aptamer biosensor system according to claim 1, characterized in that: The detection principle of the modular fluorescent RNA aptamer biosensor system is: S1: When the target circRNA is present, the P probe and the R probe are adjacent through the back-splicing site BSJ; S2: SplintR ligase catalyzes the ligation of probes to form a complete DNA template; S3: T7 RNA polymerase transcribes and generates fluorescent RNA aptamers, which emit fluorescent signals after binding to the dye; S4: No template is formed and no signal is output when there is no circRNA.

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