Current Simultaneous Discrimination of Mismatched MicroRNAs Using Base-Flipping within the α-Hemolysin Latch

The simultaneous discrimination of let-7 microRNAs (miRNAs) would greatly facilitate the early diagnosis and prognosis monitoring of diseases. In this work, a molecular beacon DNA probe was designed to be able to flip out its mismatched cytosine base within the α-hemolysin (α-HL) latch and generate completely separated blocking currents to identify the single-base difference. As a result, the characteristic blocking current of fully matched MB/let-7a and single-base mismatched MB/let-7f was 84.30 ± 0.92 and 87.05 ± 0.86% (confidence level P 95%), respectively. Let-7 miRNA family let-7a and let-7f were completely simultaneously discriminated, which could be attributed to the following strengths. (1) The statistic distribution of blocking current is extremely concentrated with a small relative standard deviation (RSD) of less than 1% and a narrow distribution range. (2) Complete separation is achieved with a high separation resolution of 1.54. (3) The cytosine base flipping out within the α-HL latch provides a universal labeling-free strategy to simultaneously discriminate the single-base mismatch. Overall, the target let-7f sequences were detected with a linear range from 0.001 to 10 pM in human serum samples containing 200 nM let-7a. Great potential has been demonstrated for precise detection, early diagnosis, and prognosis monitoring of diseases related to single-base difference.