Rapid, Sensitive, and Label-Free Detection of Long Noncoding RNAs in Breast Cancer Tissues by RecJf Exonuclease-Assisted Recombinase Polymerase Amplification.

Abstract

An abnormal expression level of long noncoding RNAs (lncRNAs) is implicated in multiple cancers, and their sensitive and rapid measurement is pivotal for early cancer diagnosis and cancer treatment. The conventional lncRNA assays often suffer from labor-intensive/time-consuming procedures and limited sensitivity. Herein, we report a simple and sensitive fluorescent biosensor for rapid and label-free measurement of lncRNAs based on recombinase polymerase amplification (RPA) without the involvement of thermal cycling and reverse transcription. Target lncRNAs can bind with the 5'-end of the DNA template to create a DNA-lncRNA hybrid, protecting the DNA template from RecJf exonuclease-mediated degradation. Subsequently, the primers hybridize with the intact DNA templates and are extended to generate the dsDNA products with the assistance of polymerase. The resultant dsDNA products may be amplified by exponential recombinase polymerase amplification to produce abundant dsDNAs, generating a distinct fluorescence signal within 10 min. This biosensor achieves a wide dynamic range from 10-17 to 10-9 M and high sensitivity with a detection limit of 1.23 aM. Moreover, it can distinguish the expressions of lncRNA HOTAIR in the tissues of healthy individuals and breast cancer patients, with broad application prospects in lncRNA-related research and early diagnosis of cancers.