A Sensitive Fluorescence Platform For CircRNA Detection Using Tetrahedral Polymerization Catalyzes Three-hairpin Assembly

Background&ObjectiveCircular RNA(circRNA)is a special kind of noncoding closed RNA,which can be used to regulate gene expression RNA in mammals.CircRNA is connected by reverse splicing,and the downstream 5’ and upstream 3’ terminal splicing sites.The cyclic ring has high tolerance to nucleic acid exonuclease.Compared with its linear counterpart,circRNA is more stable,more conservative and has a longer half-life.They have a variety of functions,including combining with microRNA(miRNA),playing the role of miRNA sponge,adsorbing miRNA,effectively inhibiting the combination of mirna and untranslated region of target gene,thus realizing the regulation of target gene.Therefore,they have unique advantages as potential markers of cancer and development targets of therapeutic strategies.It can be used in early clinical screening,clinical treatment,curative effect judgment,drug target and recurrence monitoring,etc.It can provide a diagnostic index for early diagnosis and treatment of tumors and has potential clinical application value.Compared with the conventional tumor diagnosis technology,circular RNA has the advantages of non-invasive,simple,repeatable sampling for detection and convenient dynamic monitoring.However,at present,the methods for detecting circRNA still rely on traditional northern blot,RNA sequencing technology(RNA-seq)and reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR),however,these methods have some defects in different degrees,such as low sensitivity,long time-consuming,expensive instruments,complicated operation and high false positive and negative rate,which hinders their wide application in clinic.Therefore,it is significance to develop a simple,sensitive method for detecting circRNA.In order to solve these problems,we proposed A A Sensitive Fluorescence Platform for circRNA detection Using Tetrahedral polymerization catalyzes three-hairpin assembly.Tetrahedral polymerization catalyzes three-hairpin assembly is a simple and easy-to-control nucleic acid isothermal amplification technique,which converts chemical input into amplified detectable signals by using programmed assembly reactions between nucleic acid substrates.Compared with the traditional unit point assembly technology,efficiency of the polymerized DNA tetrahedral catalytic hairpin assembly is higher,and its mechanisms are as follows:Firstly,three CHA hairpins are fixed by DNA tetrahedron to form a TDN structure.Because the tetrahedron is simple and efficient to assemble,and has a rigid structure,it can well control the distance of CHA hairpins,thus easily adjusting the effect of CHA system localization.When TDN encounters the target Circ-RNA,it is mediated by the foothold on H1,which turns H1 into a linear structure that hybridizes with the target part.The unhybridized part binds to the foothold of H2 and opens the H2 stem loop to further open the H3 hairpin to form a triggered TDN structure.There are two free viscous ends in the triggered TDN structure,which causes multiple triggered TDN to produce polymerization,which further improves the signal intensity.Finally,we apply this technique to the detection of circRNA.Therefore,this method achieves multiple amplification of the signal,improves the sensitivity and efficiency of the detection system,and constructs a new sensitive method for detecting Circ-RNA in exosomes.Methods1.The establishment of the Tetrahedral polymerization catalyzes three-hairpin assembly1.1 The required nucleic acid sequences in the system were designed,and the secondary structures of the three hairpins were predicted by NUPACK nucleic acid analysis website,and the addition of nucleic acids with tetrahedral structure was used to observe the occurrence of non-specific hybridization and to verify the loading performance of DNA tetrahedron.1.2 PAGE was used to verify the effective establishment of DNA tetrahedron and tetrahedral polymerization catalyzed three hairpin assembly system,and the successful activation of TDN after adding the target was also needed.1.3 The fluorescence platform analyzes the feasibility of the biosensor based on the self-assembly of hairpin catalyzed by polymeric DNA tetrahedron,and proves that DNA tetrahedron does have localization effect and can generate stronger signals.The fluorescence intensity and catalytic efficiency of TDN were compared with that of traditional CHA by selecting the labeled fluorescent group FAM and quenching group BHQ1 of H1.2.Performance assessment and practical application ability evaluation of Tetrahedral polymerization catalyzes three-hairpin assembly for circRNA detection2.1 Optimization of temperature and spacing for three-hairpin assembly catalyzed by tetrahedral polymerization.With the signal-to-noise ratio as the evaluation index,the optimal reaction condition was used for the subsequent experiments.2.2 Under the above optimal reaction conditions,the following target sequences at different concentrations were detected:0.1 pM,1pM,10pM,100pM,1nM,10nM,20nM,50nM,and 100nM.2.3 Detect single-base mismatching target sequence,double-base mismatching target sequence and other homologous Circ-RNA,and compare the fluorescence signal strengths among them to verify the specificity of the established detection method..ResultsA fluorescent biosensor based on Tetrahedral polymerization catalyzes three-hairpin assembly was constructed for the detection of circular RNA.Only when the target Circ-RNA is added,mediated by the foothold on the H1,the H1 becomes a linear structure that hybridizes with the target part.The unhybridized part combines with the foothold of H2 and opens the H2 stem ring to further open the H3 hairpin to form a triggered completed TDN structure,thus amplifying the signal.There are two free sticky ends in the triggered completed TDN structure,which causes multiple triggered completed TDN to produce polymerization.The intensity of the signal is further improved.Therefore,the catalytic efficiency is obviously better than the traditional catalytic hairpin assembly reaction.Under the optimal reaction conditions,firstly,the sensitivity was explored,and the fluorescence intensity was measured under the target concentration from 0.1pM to 100nM.There was a good correlation between the fluorescence intensity and the target concentration.Regression analysis showed that the signal showed a Y=6.562LogC+41.681 correlation,R2=0.968,with a good linear output range.In addition,the specificity was explored with single base mismatch target,double base mismatch target and two kinds of homologous Circ-RNA.The fluorescence results show that the mismatched target signal decreases greatly and has a good discrimination with the correct target,and other Circ-RNA signals of the same origin are lower,which will not cause misjudgment of the result.Finally,through the detection of plasma samples from healthy people and patients with liver cancer,the final result is that the sensor can make a good distinction between patients with liver cancer and healthy people.ConclusionIn this work,we established a fluorescent biosensor based on Tetrahedral polymerization catalyzes three-hairpin assembly,which can be rapid,simple,enzyme-free and highly sensitive fluorescence detection of circRNA.The platform has been preliminarily verified in clinical specimens.The biosensor strategy has great application value and potential in biological analysis and clinical diagnosis.