Specific And Sensitive Detection Of CircRNA Based On Netlike Hybridization Chain Reaction

Background: Circular RNA(circRNA)is an emerging endogenous non-coding RNA(nc RNA)with a covalently closed circular structure,which is produced by a special back-splicing process.It plays an important role in the occurrence and development of various diseases.Several studies had shown that circRNA participated in the proliferation,invasion,and metastasis of tumor cells through multiple mechanisms.The aberrant expression of circRNA can be used as a promising biomarker for cancer diagnosis and prognosis,and it is more important than previously recognized.However,the limitations of detection methods in recent years have severely restricted the related research of circRNA.Therefore,sensitive and specific detection of circRNA is of great significance for understanding the biological functions of circRNA and tracking the disease process.Aims: This study aims to develop a specific and sensitive detection of circRNA based on netlike hybridization chain reaction(net-HCR),which can detect target circRNAs in the complex environment of biological samples without the interference of linear isomers.Principle: Here,we have developed an effective circRNA detection method based on the thermostatic netlike hybridization chain reaction(HCR).It combines reverse transcription-rolling circle amplification(RTRCA)with well-designed netlike HCR to achieve dual selection and dual signal amplification.The special advantage of the unique circular structure of circRNA enables continuous rolling circle amplification,and obtains long-chain complementary DNA(c DNA)with multiple sets of the fulllength cyclic consensus sequence(CCS),realizing the first selection and amplification.Long-chain c DNAs with multiple sets of repeat sequences can trigger the subsequent net-HCR to achieve a second amplification of the signal.The net-HCR consists of a hairpin trigger probe(Trigger)and two fuel probes(H1,H2).After the Trigger recognizes the target sequence,it will initiate the continuous self-assembly of the fuel probe,and finally form a double-stranded network structure with the c DNA strand containing multiple sets of repetitive sequences,resulting in an enhanced fluorescent signal.Methods:(1)First,the performance of net-HCR in detecting c DNA strands with repetitive sequences was evaluated.The feasibility of netHCR was verified and the reaction conditions were optimized.The analytical performance of the net-HCR system is investigated under optimal conditions.(2)Next,circRNAs were specifically detected with a net-HCR-based strategy.A circRNA model was constructed,and longchain c DNAs with multiple sets of CCS were obtained by RT-RCA.The product c DNA after reverse transcription of circRNA was detected by netHCR.(3)Finally,different cell lines were selected,and tumor tissue and normal liver tissue samples from patients with liver cancer were collected to detect the expression of circ TEX 2.Results:(1)It was verified that the three probes of net-HCR could coexist stably in the buffer and self-assemble in the presence of the target gene strand by various methods.We determined that the optimal probe concentrations were Trigger 0.05 μM,H1 and H2 0.1 μM;the optimal reaction temperature was 30 ℃;and the reaction time was 3 h.Under optimal conditions,net-HCR has a linear range of 1.0 p M to 1.0 n M with a limit of detection(LOD)of 1.12 p M(S/N = 3),which can identify onebase mismatches.And the net-HCR system has a higher response value for long c DNA strands containing multiple sets of repeat sequences.(2)According to the back-spliced junction(BSJ)sequence of circ TEX 2,a small circRNA with a length of 60 nt was constructed and purified.The first amplification was achieved by RT-RCA.During this process,neither the corresponding linear RNA isoform nor the end-to-end linear RNA isoform can undergo continuous reverse transcription.The product c DNA was detected by net-HCR,and the obtained fluorescence signal was positively correlated with the circRNA concentration.(3)The expression of circ TEX 2 was detected in different cell lines and tissue samples.The results showed that circ TEX 2 was significantly down-regulated in liver cancer cell lines and tissues,which was consistent with the q PCR results.Conclusion: The circRNA-specific detection method based on netHCR can overcome the complex detection environment of biological samples without RNase R enrichment,and detect circRNAs with extremely low expression levels from biological samples such as cells and tissues.The advantage of this method is that it can exclude the interference of linear RNA isoforms and end-to-end linear RNA isoforms,and shows excellent selectivity.The fluorescence detection method does not require complicated pre-preparation,nor does it require expensive temperature control instruments under constant temperature conditions,and the operation is simpler.This strategy is of great significance for in-depth understanding of the relationship between circRNAs and various diseases.On the other hand,net-HCR also has potential applications for the detection of multiple genes containing repetitive sequences,such as telomeric DNA.