CircRNA detection method based on Argonaute protein and rolling circle amplification

Abstract

The invention discloses a circRNA (Ribonucleic Acid) detection method based on an Argonaute protein and rolling circle amplification. The method comprises the following steps: mixing a to-be-detected sample containing target circRNA with a specific reverse transcription primer and reverse transcriptase, and reacting to obtain long-chain cDNA (complementary deoxyribonucleic acid); mixing the obtained long-chain cDNA with pfAgo, gDNA and a DNA nucleic acid probe for reaction, and calculating the content of the target circRNA according to a fluorescence signal of a reaction mixed solution; the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by circRNA reverse transcription, the sequence of the DNA nucleic acid probe is specifically complementary to the sequence of the cDNA subjected to primary cutting by pfAgo, and the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent label. According to the invention, high-sensitivity, high-specificity and multi-target simultaneous analysis and detection of potential liver cancer diagnosis biomarkers such as hsacirc0001445, hsacirc0001141 and the like can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a circRNA detection method based on Argonaute protein and rolling circle amplification. Background Art

[0002] Circular RNA (circRNA) is a single-stranded non-coding RNA with a covalently closed ring structure. It can play a variety of biological functions and is associated with a variety of diseases such as cancer, neurological diseases, diabetes and atherosclerosis. Abnormally expressed circRNA is a potential therapeutic target for acute promyelocytic leukemia and can serve as a potential diagnostic and prognostic biomarker for diseases such as Alzheimer's disease, diabetes and cancer. For example, hsa_circ_0001445 and hsa_circ_0001141 are lowly expressed in liver cancer and are potential diagnostic biomarkers for liver cancer. In addition, the occurrence and 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, the development of sensitive, specific, and multi-target circRNA detection methods is of great significance for the study of the physiological and pathological functions of circRNAs and disease diagnosis and treatment.

[0003] Traditional molecular biological methods for detecting circRNA include northern blotting, RNA sequencing, fluorescence in situ hybridization (FISH), and real-time fluorescence quantitative PCR (RT-qPCR). Northern blotting can be used to distinguish circular RNA from linear RNA, but it has low sensitivity and is time-consuming. RNA sequencing is widely used in circRNA research, but it is expensive and requires professional experimental personnel, which limits its application in routine diagnosis. FISH can detect circRNA in situ, but it requires advanced microscopes to obtain fluorescent signals. RT-qPCR is currently the most commonly used method for detecting circRNA, which can achieve sensitive detection of circRNA. However, since RT-qPCR is based on the back-splicing junction (BSJ), this method cannot distinguish between trans-spliced ​​linear RNA (tsRNA) and cis-spliced ​​circRNA. In recent years, researchers have developed various new strategies for circRNA detection based on signal amplification technology, such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), and signal amplification assisted by double-stranded specific nuclease (DSN). LAMP technology can help achieve exponential amplification of circRNA in a short time, with strong specificity and high sensitivity. However, considering the fidelity of primer extension amplification catalyzed by DNA polymerase, it is uncertain whether this method can distinguish base mismatches. The circRNA detection method based on DSN can directly obtain amplified fluorescent signals, but it cannot directly detect circRNA from complex biological samples without RNase R treatment. The detection of circRNA using RCA can be carried out under constant temperature conditions, is easy to operate, and has stable signal output, but its signal amplification factor still cannot meet the requirements of clinical application.

[0004] The clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) system and Argonaute protein (Ago) are both derived from the microbial defense system. 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 the high cost and easy degradation of guide RNA; the detection target is restricted by the protospacer adjacent motif (PAM) or protospacer flanking sequence (PFS) sequence; and it is difficult to achieve multiple detection. Similar to Cas, Ago is also a programmable nuclease. Unlike Cas, Ago can recognize its target through base pairing of 5' phosphorylated guide DNA (gDNA), without the need for the presence of PAM / PFS sequence in the target. Argonaute (pfAgo) from the extremely thermophilic bacterium Pyrococcus furiosus is a prokaryotic Argonaute that can cut single-stranded DNA (ssDNA) under the guidance of gDNA. The short ssDNA produced by the cutting can also bind to pfAgo and perform a second cutting of the downstream target DNA, and is widely used in molecular diagnosis. Ago-based biosensors have many advantages such as high stability, low cost, and easy multiplex detection. However, the directional cutting of Ago is less efficient than the trans-cutting of Cas, and signal amplification is required to improve the detection sensitivity.

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

[0006] In order to solve the problems in the prior art, the present 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] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The circRNA detection method based on Argonaute protein and rolling circle amplification provided by the present invention comprises the following steps:

[0009] 1) Designing a specific reverse transcription primer that is specifically complementary to the circRNA to be tested; using the specific reverse transcription primer to perform reverse transcription rolling circle amplification on the target circRNA in the sample to be tested to obtain a long single-stranded cDNA with multiple groups of repeated sequences;

[0010] 2) adding pfAgo protein, gDNA and DNA nucleic acid probe to the system obtained in step 1) to mix and react, and calculating the content of the target circRNA according to the fluorescence signal of the reaction mixture; wherein the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of the circRNA, the DNA nucleic acid probe sequence is specifically complementary to the sequence of the cDNA after one cutting by the pfAgo protein, and the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent label.

[0011] Step 1) is specifically as follows: the target circRNA to be detected is mixed with a specific reverse transcription primer and reverse transcriptase, reacted at 42°C for 2h, and then treated at 85°C for 5s to obtain a long-chain cDNA.

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

[0013] The present invention also provides a circRNA multiplex detection system 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;

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

[0016] The beneficial technical effect of the present invention is that the present invention combines Argonaute protein and rolling circle amplification technology for the first time to construct a new method that can be used for multiple circRNA detection. The application of rolling circle amplification amplifies the detection signal and improves the detection sensitivity on the one hand, and eliminates the interference of linear RNA on the other hand; the application of Argonaute protein can further ensure the specificity of detection and realize one-pot multiple target detection by specifically recognizing the repetitive sequence in the circRNA rolling circle amplification product. Therefore, the circRNA detection method provided by the present invention realizes high sensitivity, high specificity and simultaneous analysis of multiple targets of hsa_circ_0001445 and hsa_circ_0001141. The circRNA detection method provided by the present invention also has the advantages of flexible design, low cost, wide detection range, etc., and can enrich the existing Argonaute sensing system. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Schematic diagram of the principle of the circRNA detection method based on Argonaute protein and rolling circle amplification of the present invention;

[0018] Figure 2 :Feasibility study of RT-RCA-Argonaute detection of circRNA, Figure A is the result of hsa_circ_0001445 detection signal, Figure B is the result of hsa_circ_0001141 detection signal;

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

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

[0021] Figure 5 : Detection results of circRNA expression levels in different cells, Figure A is the result of RT-RCA-Argonaute detection of hsa_circ_0001445 and hsa_circ_0001141 of the present invention, and Figure B is the result of real-time quantitative reverse transcription PCR detection of hsa_circ_0001445 and hsa_circ_0001141. DETAILED DESCRIPTION

[0022] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0023] In the present invention, a specific reverse transcription primer sequence is obtained from the target circRNA sequence. First, a specific primer is used to reverse transcribe the circRNA. Due to the circular structure of the circRNA, rolling circle amplification will produce long single-stranded cDNA with multiple sets of repeating sequences. Then, a 5'-phosphorylated gDNA that can specifically complement the long single-stranded cDNA sequence is designed and used. Under the guidance of the gDNA, pfAgo can be activated and cut the cDNA to produce a large amount of 5'-phosphorylated ssDNA. These ssDNAs can be used as gDNA for pfAgo to perform a second cut. Next, a fluorescently labeled DNA nucleic acid probe that can specifically complement the sequence of the cDNA after being cut by pfAgo once is designed and used. The second cutting of pfAgo causes the separation of the fluorescent group and the quenching group of the DNA nucleic acid probe, thereby generating a fluorescent signal. Since the DNA nucleic acid probe is designed according to the circRNA sequence, multiple detections of circRNA can be achieved by using DNA nucleic acid probes with different fluorescent labels. The schematic diagram of the principle of the circRNA detection method based on Argonaute protein and rolling circle amplification of the present invention is shown in the figure below. Figure 1 shown.

[0024] This embodiment provides a circRNA detection method based on Argonaute protein and rolling circle amplification, comprising the steps of:

[0025] S1. Providing a circRNA detection system, wherein the circRNA detection system comprises a specific reverse transcription primer, a reverse transcriptase, gDNA, a pfAgo protein and a DNA nucleic acid probe;

[0026] S2, the target circRNA to be detected is mixed with a specific reverse transcription primer and a reverse transcriptase to react to obtain a long-chain cDNA; the obtained long-chain cDNA is mixed with pfAgo, gDNA and a DNA nucleic acid probe to react, 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 the cDNA after one cut by pfAgo; the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent label;

[0027] S3. Calculate the content of the target circRNA according to the fluorescence signal of the reaction mixture.

[0028] Wherein, the circRNA detection method is for non-diagnostic purposes.

[0029] The target circRNA to be detected in the present invention is one or both of hsa_circ_0001445 or hsa_circ_0001141. Wherein, 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. In order to more conveniently analyze the feasibility and detection performance of the detection method of the present invention for detecting circRNA, in Examples 1-3, two 60nt circRNAs (circRNA1445 and circRNA1141) containing the reverse splicing sites of hsa_circ_0001445 or hsa_circ_0001141 were artificially synthesized for research, wherein the circRNA1445 sequence is shown in SEQ ID NO.3, and the circRNA1141 sequence 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 labels at both ends of the DNA probe can be arbitrarily combined from the above labels, or other suitable fluorescent labels can be used. More specifically, the 5' end of the DNA nucleic acid probe used to detect hsa_circ_0001445 is labeled with FAM, and the 3' end is labeled with BHQ1; the 5' end of the DNA nucleic acid probe used to detect hsa_circ_0001141 is labeled with Cy3, and the 3' end is labeled with BHQ2.

[0031] The present invention provides a circRNA detection system based on Argonaute protein and rolling circle amplification: the detection system comprises a specific reverse transcription primer, a reverse transcriptase, gDNA, a pfAgo protein, a DNA nucleic acid probe and a buffer; the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of the circRNA, the DNA nucleic acid probe sequence is specifically complementary to the sequence of the cDNA after one-time cutting by pfAgo, and the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent marker.

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

[0033] When the target circRNA is hsa_circ_0001445 and hsa_circ_0001141, the above-mentioned specific reverse transcription primer, gDNA and DNA nucleic acid probe are also included. 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] Simultaneous detection of hsa_circ_0001445 and hsa_circ_0001141 by reverse transcription rolling circle amplification combined with pfAgo cleavage. This example describes a method for multiple circRNA detection using pfAgo enzyme cleavage activity after amplifying circRNA using reverse transcription rolling circle amplification technology.

[0037] Reverse transcription rolling circle amplification reaction: HiScriptⅢ1st Strand cDNA Synthesis Kit (+gDNAwiper) was used for the operation: 2.5μl of circRNA1445 and circRNA1141 and 1μl 10×RT Mix, 1μl Enzyme Mix, 0.25μl of 20μM reverse transcription primers RT1445 and RT1141 and 1.5μl RNase-free water were mixed and reacted at 42℃ for 2h, and then treated at 85℃ for 5s to obtain long-chain cDNA.

[0038] pfAgo enzyme digestion reaction: Take 2 μl of the mixture obtained in the above reaction, mix it with 2 μl 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 RNase-free water, and react at 95°C for 30 min.

[0039] In order to investigate the feasibility of this method for multiplex detection of circRNA, 5 groups of experiments were designed for verification, and the concentrations of circRNA1445 and circRNA1141 used were 500nM. The first group was set up without circRNA1445 and circRNA1141, the second group was set up without circRNA1141, the third group was set up without circRNA1445, the fourth group was set up as a normal experimental group, and the fifth group was set up with reaction buffer to replace circRNA1445 and circRNA1141. After the reverse transcription rolling circle amplification and pfAgo enzyme digestion reaction, the fluorescence spectrum was measured. The results are shown in Figure 2 As shown, when circRNA1445 and circRNA1141 were present, obvious FAM and CY3 fluorescence signals were detected at 521 nm and 566 nm, respectively. The results showed that this method can be used to detect multiple circRNAs simultaneously.

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

[0041] In order to investigate the specificity of this method for multiplex detection of circRNA, 7 groups of experiments were designed for verification, and the concentrations of circRNA1445 and circRNA1141 used were 500nM. Group 1 was set as no circRNA1445 and circRNA1141, Group 2 was set as no circRNA1141, Group 3 was set as no circRNA1445, Group 4 was set as the normal experimental group, Group 5 was set as other circRNA, Group 6 was set as other miRNA, and Group 7 was set as circRNA replaced with reaction buffer. After the reverse transcription rolling circle amplification and pfAgo enzyme digestion reaction, the FAM and CY3 fluorescence signal intensities detected at 521nm and 566nm were measured respectively. The results are shown in Figure 2. Figure 3 As shown in the figure, there is an obvious fluorescence signal only in the presence of the corresponding circRNA. The results show that this method can be used to specifically detect circRNA.

[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 at 0-10 nM, and the FAM and CY3 fluorescence signal intensities detected at 521 nm and 566 nm were measured after the reverse transcription rolling circle amplification and pfAgo enzyme digestion reactions. Figure 4 As shown, the linear range of detection is 100fM-10nM, and the detection limits of circRNA1445 and circRNA1141 calculated by the 3σ / s method are 1.56fM and 1.69fM, respectively. The results show that this method can be used for the simultaneous sensitive detection of circRNA1445 and circRNA1141.

[0044] In Examples 1-3, circRNA1445 and circRNA1141 were studied. Since they contain the reverse splicing site of hsa_circ_0001445 or hsa_circ_0001141, the results of Examples 1-3 are also applicable to the detection of the actual target hsa_circ_0001445 or hsa_circ_0001141.

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

[0046] In order to explore the feasibility of this method for quantitative detection of circRNA in actual samples, 1-5ug RNA samples were extracted from normal human liver cells (LO2) and human liver cancer cells (Hep3B). The expression levels of hsa_circ_0001445 and hsa_circ_0001141 in the two samples were detected using the detection method of the present invention and real-time quantitative reverse transcription PCR, respectively. The results are shown in Figure 2. Figure 5 The results showed that the fluorescence intensity of hsa_circ_0001445 and hsa_circ_0001141 detected by the detection method of the present invention in Hep3B cells was lower than LO2, indicating that the content of hsa_circ_0001445 and hsa_circ_0001141 in Hep3B cells was relatively low, and the detection results were consistent with those of real-time quantitative reverse transcription PCR (where 1445-QF, 1445-QR and 1141-QF, 1141-QR in Table 1 were used for hsa_circ_0001445 and hsa_circ_0001141 detection, and GAPDH-QF and GAPDH-QR in Table 1 were used for GAPDH internal reference detection). The results showed that the detection method of the present invention has the ability to accurately detect circRNA in real samples.

[0047] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A circRNA detection method based on Argonaute protein and rolling circle amplification, characterized in that: The detection method comprises the following steps: 1) Designing a specific reverse transcription primer that is specifically complementary to the circRNA to be tested; using the specific reverse transcription primer to perform reverse transcription rolling circle amplification on the target circRNA in the sample to be tested to obtain a long single-stranded cDNA with multiple groups of repeated sequences; 2) adding pfAgo protein, gDNA and DNA nucleic acid probe to the system obtained in step 1) to mix and react, and calculating the content of the target circRNA according to the fluorescence signal of the reaction mixture; wherein the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of the circRNA, the DNA nucleic acid probe sequence is specifically complementary to the sequence of the cDNA after one cutting by the pfAgo protein, and the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent label.

2. The method according to claim 1, characterized in that The target circRNA to be detected is one or both of hsa_circ_0001445 and hsa_circ_0001141.

3. The method according to claim 2, characterized in that When the target circRNA is only hsa_circ_0001445, the sequence of the specific reverse transcription primer in step 1) is shown as SEQ ID NO.5, the sequence of the gDNA in step 2) is shown as SEQ ID NO.7, and the sequence of the DNA nucleic acid probe is shown as SEQ ID NO.

9.

4. The method according to claim 2, characterized in that: When the target circRNA is only hsa_circ_0001141, the sequence of the specific reverse transcription primer in step 1) is shown as SEQ ID NO.6, the sequence of the gDNA in step 2) is shown as SEQ ID NO.8, and the sequence of the DNA nucleic acid probe is shown as SEQ ID NO.

10.

5. The method according to claim 2, characterized in that: When the target circRNA is hsa_circ_0001445 and hsa_circ_0001141, the specific reverse transcription primers in step 1) include primers shown in sequence SEQ ID NO.5 and primers shown in sequence SEQ ID NO.6 in equal proportions; the gDNA in step 2) includes gDNA shown in sequence SEQ ID NO.7 and gDNA shown in sequence SEQ ID NO.8 in equal proportions; the DNA nucleic acid probe includes a DNA nucleic acid probe shown in sequence SEQ ID NO.9 and a DNA nucleic acid probe shown in sequence SEQ ID NO.10 in equal proportions, and the two DNA nucleic acid probes have different fluorescent labels.

6. The method according to claim 1, characterized in that Step 1) is specifically as follows: the target circRNA to be detected is mixed with a specific reverse transcription primer and reverse transcriptase, reacted at 42°C for 2h, and then treated at 85°C for 5s to obtain a long-chain cDNA.

7. The method according to claim 1, characterized in that The 5' end of the DNA nucleic acid probe is labeled with FAM, Cy3, FITC, ROX, HEX or Cy5; and the 3' end is labeled with BHQ1, BHQ2 or BHQ3.

8. The method according to claim 2, characterized in that: The DNA nucleic acid probe used to detect hsa_circ_0001445 was labeled with FAM at the 5' end and with BHQ1 at the 3' end; the DNA nucleic acid probe used to detect hsa_circ_0001141 was labeled with Cy3 at the 5' end and with BHQ2 at the 3' end.

9. A circRNA multiplex detection system based on Argonaute protein and rolling circle amplification, characterized in that: The target circRNAs to be detected were 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; The specific reverse transcription primers include primers shown in SEQ ID NO.5 and primers shown in SEQ ID NO.6 in equal proportions; the gDNA includes gDNA shown in SEQ ID NO.7 and gDNA shown in SEQ ID NO.8 in equal proportions; the DNA nucleic acid probe includes a DNA nucleic acid probe shown in SEQ ID NO.9 and a DNA nucleic acid probe shown in SEQ ID NO.10 in equal proportions, the 5' end of the DNA nucleic acid probe shown in SEQ ID NO.9 is labeled with FAM and the 3' end is labeled with BHQ1; the 5' end of the DNA nucleic acid probe shown in SEQ ID NO.10 is labeled with Cy3 and the 3' end is labeled with BHQ2.

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