Hyper-dendritic rolling circle amplification for RNA and GSH detection

Abstract

An amplified and highly sensitive hyper-dendritic rolling circle amplification (HRCA) is presented herein. This HRCA system consists of only one circular nucleic acid template that contains three functional areas. One part is an identification sequence of the analyte RNA. The other part is a sequence identical to the molecular beacon modified with a fluorophore/quencher pair. The third part is a sequence identical to the loop region of the coded hairpin structure. These three sequences of circular nucleic acid template can stably co-exist until introducing an analyte RNA to trigger RCA events, which leads to self-assembly of hyper-dendritic DNA structures. We employ stem-loop-structured fluorescent molecular beacon to transduce the autonomous operation of this RCA machine to fluorescent signal. The system can easily achieve high-sensitive detection of target miRNA-21 to 10−18 M. By the design of reduced thiol-cleavable disulfide bond in another DNA sequence, which can be cleaved by the exchange reaction of thiol ligands. After cleavage, DNA is released as the linker and the HRCA principle is successfully applied to display excellent selectivity and sensitivity for reduced thiol (take GSH, for example) to 0.1 nM. In conclusion, the HRCA with high amplification efficiency has the advantages of simple, uniformity, stability and low cost. So it has a wide application prospect in the development of biosensing technology, programmable assembly of DNA architecture and biological detection.