Engineered G-Quadruplex-Embedded Self-Quenching Probes Regulate Single Probe-Based Multiplex Isothermal Amplification to Light Road Lamp Probes for Sensitized Determination of microRNAs
Jie Wang, Jiayin Sun, Jing Zhang, Chenlin Shen, Xinlei Zhang, Jianguo Xu
Abstract
Design of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics. In this work, we engineered a miRNA-21-responsive G-quadruplex-embedded self-quenching probe (GE-SQP) that can regulate single probe-based multiplex amplifications. The free GE-SQP is tightly locked in a quenching state with no active G-quadruplexes. Introduction of target miRNA to hybridize with GE-SQP would induce a multiplex isothermal amplification to significantly build a lot of one-bulb-contained road lamp probe (OC-RLP) and two-bulb-contained road lamp probe (TC-RLP) using G-quadruplex as the lamp bulb. When lightened by thioflavin T (ThT), beams of fluorescence were emitted to show the presented miRNA-21. Specially, the whole amplification is only a one probe-involved one-step reaction without any wasted species. The mix-to-detection and all-in amplification behavior allows the sensing system a maximally maintained operation simplicity and high assay performance. In such a way, the detection range of miRNA-21 is from 1 fM to 1 nM with a limit of detection of 0.86 fM. The practicability was demonstrated by determining miRNA-21 from serum samples with acceptable results. We expect that this method can open a new avenue for exploring advanced biosensors with improved analytical performances.