Method for detecting circrna based on argonaute protein and rolling circle amplification

Abstract

The application discloses a circRNA detection method based on Argonaute protein and rolling circle amplification. The method comprises the following steps: mixing a sample containing target circRNA to be detected with a specific reverse transcription primer and reverse transcriptase to obtain long-chain cDNA; mixing the obtained long-chain cDNA with pfAgo, gDNA and a DNA nucleic acid probe to carry out a reaction, and calculating the content of the target circRNA according to the fluorescence signal of the reaction mixture; the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of the circRNA; the sequence of the DNA nucleic acid probe is specifically complementary to the sequence of the cDNA after one cutting of the cDNA by the pfAgo; and the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent label. The application can realize high-sensitivity, high-specificity and multiple target simultaneous analysis and detection of potential diagnostic biomarkers of liver cancer such as hsa_circ_0001445 and hsa_circ_0001141.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a method for detecting circRNA based on Argonaute protein and rolling circle amplification. Background Technology

[0002] Circular RNA (circRNA) is a single-stranded non-coding RNA with a covalently closed circular structure. It performs a variety of biological functions and is associated with various diseases, including cancer, neurological disorders, diabetes, and atherosclerosis. Aberrantly expressed circRNAs are potential therapeutic targets for acute promyelocytic leukemia and can serve as potential diagnostic and prognostic biomarkers for diseases such as Alzheimer's disease, diabetes, and cancer. For example, hsa_circ_0001445 and hsa_circ_0001141 are expressed at low levels in liver cancer and are potential diagnostic biomarkers for this disease. Furthermore, the development of a disease is often associated with abnormal expression of multiple circRNAs, and abnormal expression of a single circRNA often occurs in multiple diseases. Therefore, developing sensitive, specific, and multitarget circRNA detection methods is of great significance for the study of the physiological and pathological functions of circRNAs and for disease diagnosis and treatment.

[0003] Traditional molecular biology methods for detecting circRNAs include Northern blotting, RNA sequencing, fluorescence in situ hybridization (FISH), and real-time quantitative PCR (RT-qPCR). Northern blotting can distinguish between circular and linear RNAs, but it has low sensitivity and is time-consuming. RNA sequencing is widely used in circRNA research, but its high cost and the need for specialized personnel limit its application in routine diagnostics. FISH enables in situ detection of circRNAs, but requires advanced microscopy to obtain fluorescence signals. RT-qPCR is currently the most commonly used method for detecting circRNAs, enabling sensitive detection. However, because RT-qPCR is based on the back-splicing junction (BSJ), it cannot distinguish between trans-spliced ​​linear RNA (tsRNA) and cis-reverse-spliced ​​circRNAs. In recent years, researchers have developed various novel strategies for circRNA detection based on signal amplification techniques, such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), and signal amplification assisted by double-stranded specific nucleases (DSNs). LAMP technology can help achieve exponential amplification of circRNAs in a short time, with high specificity and sensitivity. However, considering the fidelity of primer extension amplification catalyzed by DNA polymerase, it is uncertain whether this method can distinguish base mismatches. DSN-based circRNA detection methods can directly obtain amplified fluorescence signals, but cannot directly detect circRNAs from complex biological samples without RNase R treatment. RCA detection of circRNAs can be performed under isothermal conditions, is easy to operate, and provides stable signal output, but its signal amplification factor still cannot meet the requirements of clinical applications.

[0004] Both the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) system and the Argonaute protein (Ago) originate from microbial defense systems. The CRISPR / Cas system has been widely used in gene editing and nucleic acid detection. However, CRISPR / Cas-based biosensors still have some inherent limitations, including high cost and easy degradation of guide RNA; detection targets are restricted by protospacer adjacent motifs (PAMs) or protospacer flanking sequences (PFSs); and multiplexing is difficult. Similar to Cas, Ago is also a programmable nuclease. Unlike Cas, Ago can recognize its targets through base pairing of 5' phosphorylated guide DNA (gDNA), without requiring the presence of PAM / PFS sequences on the target. Argonaute (pfAgo), derived from the extreme thermophilic bacterium *Pyrococcus furiosus*, is a prokaryotic argonaute that can cleave single-stranded DNA (ssDNA) under gDNA guidance. The resulting short ssDNA can bind to pfAgo and perform a second cleavage of downstream target DNA, making it widely used in molecular diagnostics. Ago-based biosensors offer advantages such as high stability, low cost, and ease of multiplexing. However, Ago's directional cleavage is less efficient than Cas's trans-cleavage, requiring signal amplification to improve detection sensitivity.

[0005] Therefore, in order to overcome the technical shortcomings of existing detection methods, it is urgent to develop a circRNA detection method with high sensitivity, high specificity and the ability to simultaneously analyze multiple targets, so as to apply it to the detection of potential diagnostic biomarkers for liver cancer such as hsa_circ_0001445 and hsa_circ_0001141. Summary of the Invention

[0006] To address the problems in the existing technology, this invention proposes a circRNA detection method based on Argonaute protein and rolling circle amplification, which can achieve high sensitivity and high specificity detection of hsa_circ_0001445 and hsa_circ_0001141.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] The method for detecting circRNA based on Argonaute protein and rolling circle amplification provided by this invention includes the following steps:

[0009] 1) Design specific reverse transcription primers that are specifically complementary to the target circRNA; use the specific reverse transcription primers to perform reverse transcription rolling circle amplification of the target circRNA in the test sample to obtain long single-stranded cDNA with multiple sets of repeating sequences.

[0010] 2) Add pfAgo protein, gDNA and DNA nucleic acid probe to the system obtained in step 1) and mix to react. Calculate the content of the target circRNA based on the fluorescence signal of the reaction mixture. The nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of the circRNA, and the DNA nucleic acid probe sequence is specifically complementary to the sequence of cDNA after being cleaved once by pfAgo protein. The DNA nucleic acid probe is a fluorescently labeled single-stranded DNA probe.

[0011] Step 1) Specifically, the target circRNA to be detected is mixed with specific reverse transcription primers and reverse transcriptase, reacted at 42°C for 2 hours, and then treated at 85°C for 5 seconds to obtain long cDNA.

[0012] The 5' end of the DNA nucleic acid probe is labeled with FAM, Cy3, FITC, ROX, HEX or Cy5; the 3' end is labeled with BHQ1, BHQ2 or BHQ3.

[0013] This invention also provides a multiplex detection system for circRNA based on Argonaute protein and rolling circle amplification, wherein the target circRNAs to be detected are hsa_circ_0001445 and hsa_circ_0001141;

[0014] The multiplex detection system includes specific reverse transcription primers, reverse transcriptase, gDNA, pfAgo protein, DNA nucleic acid probes, and buffer solution;

[0015] The specific reverse transcription primers comprise primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 in equal proportions; the gDNA comprises gDNA shown in SEQ ID NO. 7 and SEQ ID NO. 8 in equal proportions; the DNA nucleic acid probes comprise DNA nucleic acid probes shown in SEQ ID NO. 9 and SEQ ID NO. 10 in equal proportions, wherein the 5' end of the DNA nucleic acid probe shown in SEQ ID NO. 9 is labeled with FAM and the 3' end with BHQ1; and the 5' end of the DNA nucleic acid probe shown in SEQ ID NO. 10 is labeled with Cy3 and the 3' end with BHQ2.

[0016] The beneficial technical effects of this invention are as follows: This invention is the first to combine Argonaute protein and rolling circle amplification (RoBAM) technology to construct a novel method for multiplex circRNA detection. The application of RoBAM amplifies the detection signal, improving detection sensitivity, while eliminating interference from linear RNA. The application of Argonaute protein, by specifically recognizing repetitive sequences in the RoBAM products of circRNA, further ensures detection specificity and enables single-pot multiplex target detection. Therefore, the circRNA detection method provided by this invention achieves high sensitivity, high specificity, and simultaneous analysis of multiple targets, including hsa_circ_0001445 and hsa_circ_0001141. The circRNA detection method provided by this invention also has advantages such as flexible design, low cost, and wide detection range, and can enrich existing Argonaute sensing systems. Attached Figure Description

[0017] Figure 1 : A schematic diagram illustrating the principle of the circRNA detection method based on Argonaute protein and rolling circle amplification in this invention;

[0018] Figure 2 Feasibility study of RT-RCA-Argonaute detection of circRNA. Figure A shows the detection signal result of hsa_circ_0001445, and Figure B shows the detection signal result of hsa_circ_0001141.

[0019] Figure 3 : RT-RCA-Argonaute detection of circRNA specificity study. Figure A shows the detection signal results of hsa_circ_0001445, and Figure B shows the detection signal results of hsa_circ_0001141.

[0020] Figure 4: Sensitivity study of circRNA detection by RT-RCA-Argonaute. Figure A shows the sensitivity results of hsa_circ_0001445, and Figure B shows the sensitivity results of hsa_circ_0001141.

[0021] Figure 5 Figure A shows the results of RT-RCA-Argonaute detection of hsa_circ_0001445 and hsa_circ_0001141 in different cells, and Figure B shows the results of real-time quantitative reverse transcription PCR detection of hsa_circ_0001445 and hsa_circ_0001141. Detailed Implementation

[0022] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.

[0023] In this invention, specific reverse transcription primer sequences are obtained from the target circRNA sequence. First, the circRNA is reverse transcribed using the specific primers. Rolling circle amplification, due to the circular structure of the circRNA, produces long single-stranded cDNA with multiple sets of repeating sequences. Then, 5' phosphorylated gDNA specifically complementary to the long single-stranded cDNA sequence is designed and used. pfAgo, guided by the gDNA, can be activated and cleave the cDNA to produce a large amount of 5' phosphorylated ssDNA. This ssDNA can be used as gDNA for pfAgo in a second cleavage. Next, fluorescently labeled DNA nucleic acid probes specifically complementary to the cDNA sequence after the first cleavage by pfAgo are designed and used. The second cleavage by pfAgo causes the separation of the fluorescent and quenching groups on the DNA nucleic acid probes, thereby generating a fluorescent signal. Since the DNA nucleic acid probes are designed based on the circRNA sequence, multiplex detection of circRNA can be achieved by using DNA nucleic acid probes with different fluorescent labels. A schematic diagram of the principle of the circRNA detection method based on Argonaute protein and rolling circle amplification is shown below. Figure 1 As shown.

[0024] This embodiment provides a method for detecting circRNA based on Argonaute protein and rolling circle amplification, including the following steps:

[0025] S1. Provide a circRNA detection system, which includes specific reverse transcription primers, reverse transcriptase, gDNA, pfAgo protein, and DNA nucleic acid probes;

[0026] S2. The target circRNA to be detected is mixed with specific reverse transcription primers and reverse transcriptase to obtain long cDNA; the obtained long cDNA is mixed with pfAgo, gDNA and DNA nucleic acid probe to obtain long cDNA; the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of circRNA, and the DNA nucleic acid probe sequence is specifically complementary to the cDNA sequence after one cleavage by pfAgo; the DNA nucleic acid probe is a fluorescently labeled single-stranded DNA probe.

[0027] S3. Calculate the content of the target circRNA based on the fluorescence signal of the reaction mixture.

[0028] The circRNA detection method described herein is not for diagnostic purposes.

[0029] The target circRNA to be detected in this invention is one or both of hsa_circ_0001445 and hsa_circ_0001141. The sequence of hsa_circ_0001445 is shown in SEQ ID NO.1, and the sequence of hsa_circ_0001141 is shown in SEQ ID NO.2. To facilitate the analysis of the feasibility and performance of the detection method for circRNA in this invention, in Examples 1-3, two artificially synthesized 60nt circRNAs (circRNA1445 and circRNA1141) containing a back-splicing site of hsa_circ_0001445 or hsa_circ_0001141 were used for research. The sequence of circRNA1445 is shown in SEQ ID NO.3, and the sequence of circRNA1141 is shown in SEQ ID NO.4.

[0030] In some embodiments, the 5' end of the fluorescently labeled single-stranded DNA probe is labeled with FAM, Cy3, FITC, ROX, HEX, or Cy5, and the 3' end is labeled with BHQ1, BHQ2, or BHQ3. The labeling at both ends of the DNA probe can be any combination of the above labels, or other suitable fluorescent labels can be used. More specifically, the DNA nucleic acid probe used to detect hsa_circ_0001445 has a FAM label at the 5' end and a BHQ1 label at the 3' end; the DNA nucleic acid probe used to detect hsa_circ_0001141 has a Cy3 label at the 5' end and a BHQ2 label at the 3' end.

[0031] This invention provides a circRNA detection system based on Argonaute protein and rolling circle amplification: the detection system includes specific reverse transcription primers, reverse transcriptase, gDNA, pfAgo protein, DNA nucleic acid probe, and buffer; the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of circRNA, the DNA nucleic acid probe sequence is specifically complementary to the sequence of cDNA after one cleavage by pfAgo, and the DNA nucleic acid probe is a fluorescently labeled single-stranded DNA probe.

[0032] When the target circRNA is only hsa_circ_0001445, the sequence of the specific reverse transcription primer in step 1) is shown in SEQ ID NO.5, the sequence of the gDNA in step 2) is shown in SEQ ID NO.7, and the sequence of the DNA nucleic acid probe is shown in SEQ ID NO.9. When the target circRNA is only hsa_circ_0001141, the sequence of the specific reverse transcription primer in step 1) is shown in SEQ ID NO.6, the sequence of the gDNA in step 2) is shown in SEQ ID NO.8, and the sequence of the DNA nucleic acid probe is shown in SEQ ID NO.10.

[0033] When the target circRNAs are hsa_circ_0001445 and hsa_circ_0001141, they simultaneously contain the aforementioned specific reverse transcription primers, gDNA, and DNA nucleic acid probes. The nucleic acid sequences used in the experiment are shown in Table 1.

[0034]

[0035] Example 1: Feasibility Analysis of the Detection Method of the Present Invention

[0036] This embodiment describes a method for simultaneous detection of hsa_circ_0001445 and hsa_circ_0001141 using pfAgo restriction enzyme activity after amplification of circRNA using reverse transcription rolling circle amplification technology.

[0037] Reverse transcription rolling circle amplification reaction: The HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper) was used: 2.5 μl each of circRNA1445 and circRNA1141 and 1 μl of 10×RT Mix, 1 μl of Enzyme Mix, 0.25 μl each of 20 μM reverse transcription primers RT1445 and RT1141 and 1.5 μl of RNase-free water were mixed and reacted at 42 °C for 2 h, and then treated at 85 °C for 5 s to obtain long cDNA.

[0038] pfAgo enzyme digestion reaction: Take 2 μl of the mixture obtained from the above reaction, mix it with 2 μl of 10×pfAgo reaction buffer, 1 μl each of 0.5 μM gDNA G1445 and G1141, 1 μl each of 1 μM DNA nucleic acid probes FQ1445 and CQ1141, and 2.5 μl of RNase-free water, and react at 95℃ for 30 min.

[0039] To investigate the feasibility of this method for multiplex detection of circRNA, five sets of experiments were designed for validation. The concentrations of circRNA1445 and circRNA1141 used were 500 nM. Group 1 (without circRNA1445 and circRNA1141), Group 2 (without circRNA1141), Group 3 (without circRNA1445), Group 4 (normal experimental group), and Group 5 (using reaction buffer to replace circRNA1445 and circRNA1141) were all performed. Fluorescence spectra were measured after reverse transcription rolling circle amplification and pfAgo restriction enzyme digestion. The results are as follows: Figure 2 As shown, when circRNA1445 and circRNA1141 were present, significant FAM and CY3 fluorescence signals were detected at 521 nm and 566 nm, respectively. These results indicate that this method can be used for the simultaneous detection of multiple circRNAs.

[0040] Example 2: Specificity analysis of the detection method of the present invention

[0041] To investigate the specificity of this method for multiplex detection of circRNAs, seven sets of experiments were designed for validation, using circRNA1445 and circRNA1141 at concentrations of 500 nM. The experimental groups were: Group 1 (without circRNA1445 and circRNA1141), Group 2 (without circRNA1141), Group 3 (without circRNA1445), Group 4 (normal experimental group), Group 5 (with other circRNAs), Group 6 (with other miRNAs), and Group 7 (with circRNAs replaced by reaction buffer). After reverse transcription rolling circle amplification and pfAgo enzyme digestion, the fluorescence signal intensities of FAM and CY3 detected at 521 nm and 566 nm, respectively, were measured. The results are as follows: Figure 3 As shown, a significant fluorescence signal is only observed in the presence of the corresponding circRNA. These results indicate that this method can be used for the specific detection of circRNAs.

[0042] Example 3: Sensitivity Analysis of the Detection Method of the Present Invention

[0043] To determine the sensitivity of this method for multiplex detection of circRNA, the final concentrations of circRNA1445 and circRNA1141 were set to 0-10 nM. After reverse transcription rolling circle amplification and pfAgo digestion, the fluorescence signal intensities of FAM and CY3 detected at 521 nm and 566 nm, respectively, were measured. The results are as follows: Figure 4 As shown, the linear detection range is 100 fM–10 nM. The detection limits for circRNA1445 and circRNA1141, calculated using the 3σ / s method, are 1.56 fM and 1.69 fM, respectively. These results indicate that this method can be used for the simultaneous and sensitive detection of circRNA1445 and circRNA1141.

[0044] Examples 1-3 were conducted using circRNA1445 and circRNA1141 as subjects. Since they contain the reverse splicing sites of hsa_circ_0001445 or hsa_circ_0001141, the results of Examples 1-3 are also applicable to the detection of the actual targets hsa_circ_0001445 or hsa_circ_0001141.

[0045] Example 4: Detection of actual samples using the detection method of the present invention.

[0046] To explore the feasibility of this method for quantitative detection of circRNA in actual samples, 1-5 μg RNA samples were extracted from normal human hepatocytes (LO2) and human hepatocellular carcinoma cells (Hep3B). The expression levels of hsa_circ_0001445 and hsa_circ_0001141 in the two samples were detected using the detection method of this invention and real-time quantitative reverse transcription PCR, respectively. The results are as follows: Figure 5 The results showed that the fluorescence intensity of hsa_circ_0001445 and hsa_circ_0001141 detected by the method of the present invention was lower than LO2 in Hep3B cells, indicating that the content of hsa_circ_0001445 and hsa_circ_0001141 in Hep3B cells was relatively low. This result showed good consistency with the detection results of real-time quantitative reverse transcription PCR (where hsa_circ_0001445 and hsa_circ_0001141 were detected using 1445-QF, 1445-QR, 1141-QF, and 1141-QR in Table 1, and GAPDH-QF and GAPDH-QR were used as internal references for GAPDH detection in Table 1). The results indicate that the detection method of the present invention has the ability to accurately detect circRNA in real samples.

[0047] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A multiplex detection system for circRNA based on Argonaute protein and rolling circle amplification, characterized in that, The target circRNAs to be detected are hsa_circ_0001445 and hsa_circ_0001141; The multiplex detection system includes specific reverse transcription primers, reverse transcriptase, gDNA, pfAgo protein, DNA nucleic acid probes, and buffer solution; The specific reverse transcription primers comprise primers with nucleotide sequences as shown in SEQ ID NO. 5 and primers with nucleotide sequences as shown in SEQ ID NO. 6 in equal proportions; the gDNA comprises gDNA with nucleotide sequences as shown in SEQ ID NO. 7 and gDNA with nucleotide sequences as shown in SEQ ID NO. 8 in equal proportions; the DNA nucleic acid probes comprise DNA nucleic acid probes with nucleotide sequences as shown in SEQ ID NO. 9 and DNA nucleic acid probes with nucleotide sequences as shown in SEQ ID NO. 10 in equal proportions, with the two DNA nucleic acid probes having different fluorescent labels.

2. The circRNA multiplex detection system based on Argonaute protein and rolling circle amplification according to claim 1, characterized in that, The DNA nucleic acid probe shown in SEQ ID NO. 9 has a FAM label at its 5' end and a BHQ1 label at its 3' end; the DNA nucleic acid probe shown in SEQ ID NO. 10 has a Cy3 label at its 5' end and a BHQ2 label at its 3' end.

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