Ultrasensitive Detection of RNA with Single-Base Resolution by Coupling Electrochemical Sensing Strategy with Chimeric DNA Probe-Aided Ligase Chain Reaction

Accurate and sensitive detection of single-base mutations in RNAs is of great value in basic studies of life science and medical diagnostics. However, the current available RNA detection methods are challenged by heterogeneous clinical samples in which trace RNA mutants usually existed in a large pool of normal wild sequences. Thus, there is still great need for developing the highly sensitive and highly specific methods in detecting single-base mutations of RNAs in heterogeneous clinical samples. In the present study, a new chimeric DNA probe-aided ligase chain reaction-based electrochemical method (cmDNA-eLCR) was developed for RNA mutation detection through the BSA-based carrier platform and the horseradish peroxidase-hydrogen peroxide-tetramethylbenzidine (HRP-H₂O₂-TMB) system. The denaturing polyacrylamide gel electrophoresis and a fluorophore-labeled probe was ingeniously designed to demonstrate the advantage of cmDNA in ligation to normal DNA templated by RNA with the catalysis of T4 RNA ligase 2 as well as its higher selectivity than DNA ligase system. Finally, the proposed cmDNA-eLCR, compared with the traditional eLCR, showed excellent performance in discriminating single base-mismatched sequences, where the signal response for mismatched targets at a high concentration could overlap completely with that for the blank control. Besides, this cmDNA-eLCR assay had a wide linear range crossing six orders of magnitude from 1.0 × 10^-15 to1.0 × 10^-10 M with a limit of detection as low as 0.6 fM. Furthermore, this assay was applied to detect RNA in real sample with a satisfactory result, thereby demonstrating its great potential in diagnosis of RNA-related diseases.