Research On Nucleic Acid Biosensing Based On Toehold Mediated Strand Displacement And Rolling Circle Amplification

Objective: Nucleic acid Detection is essential for the good practice of precision medicine.Toehold exchange and toehold displacement are two most widely used strategies in nucleic acid analysis because of their supposed high specificity.However,as far as we known,there was no direct comparison between the two strategies to decide which one is more specific for nucleic acid analysis.rolling circle amplification(RCA)has attracted great attention in nucleic acid analysis for its simplicity,robustness and high amplification power.In this research,Firstly,a systematic comparison of the specificity of SNVs detection based on toehold exchange and toehold displacement was performed to obtain more specific strategies for nucleic acid detection.Then,experiment to improve the efficiency of toehold exchange through breaking the equilibrium of the reaction was conducted.Lastly,Coupled toehold exchange and RCA were used to explore a highly specific and sensitive method for nucleic acid detection,and a more versatile method for RNA detection was established through the combination of toehold exchange and RCA.Methods: we firstly systematically compared the performance of the SNVs detection based on "toehold exchange" and "toehold displacement" in three different buffers(20 mM Tris-HCL,1× PBS,1× TAE)and analyzed the discrimination factor(DF,which represent the discrimination ability)of the above two strategies from both theoretical and experimental results.Then,"plug the reaction" which was achieved by adding "plugging probe" at the equilibrium of toehold exchange was proposed and demonstrated.The plugging probe reacted with the product of toehold exchange,thus breaking the equilibrium of the reaction and successfully amplified the signal.Taking miRNA let-7d as a model target,A fluorescence biosensing for specific discrimination and universal signal amplification of RNA detection was developed by coupling toehold exchange with RCA through nucleolytic conversion of a structure-switched hairpin probe.Results: Toehold exchange showed a higher specificity compared with toehold displacement.The experimental DF value of toehold exchange is about 6 and that of toehold displacement is about 1.It showed that the signal can be amplified 2-3 times through the principle of "plug the reaction" without the sacrifice of specificity.Taking miRNA let-7d as a model target,the developed RNA biosensing showed a wide linear range from 1 fM to 1 nM with a detection limit of 0.46 fM and exhibited a good selectivity even in distinguishing homologous miRNAs with 2-nt or 3-nt difference.Conclusion: Toehold exchange exhibited a higher discrimination than toehold displacement from both experimentally and theoretically.It will provide universal guidelines for nucleic acid detection that is required high specificity.The principle of "plug the reaction" will present new avenues for sensitive nucleic acid analysis.The fluorescence biosensing for RNA detection based on toehold exchange and RCA showed good detection performance and can be used for other RNA detection only by changing the sequence of detection probe without redesigning the sequence of RCA template.