Triple ligation-based formation of a G-quadruplex for simultaneous detection of multiple miRNAs

The simultaneous detection of multiple microRNAs (miRNA) is of great necessity but has not been extensively studied. This prompted our study, which involved the development of a triple ligation-based system for detecting three miRNAs at the same time. We designed a multi-ligation-padlock (MLP) probe that consists of three parts, each of which is complementary to two different miRNAs at the same time. In the presence of all three miRNAs, the probe becomes circularized, but in the absence of even one target, the probe remains linear. The first part of the MLP probe (MLP1) contains a T7 promoter part that can initiate RNA synthesis for any given target condition. However, it also includes a G-quadruplex complementary segment, which can only form a parallel RNA G-quadruplex through rolling circle transcription by the circularized template in the presence of all three targets. In this case, the application of our parallel G-quadruplex sensing fluorescent probe lutidine DESA (LutD) produces a strong signal. However, in the absence of any one of the targets, the RNA G-quadruplex cannot be formed and ultimately the LutD probe does not generate any signal. This difference in the signal intensity represents the presence or absence of all the target miRNAs. With our system, we were able to detect miRNA 21 at levels as low as 1.13 fM, miRNA 146a as low as 1.37 fM, and miRNA 25b as low as 1.51 fM within 45 minutes, confirming that our novel system can selectively and sensitively diagnose triple miRNAs.