Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues

Abstract

Circular RNAs (circRNAs) as endogenous non-coding RNAs are characterized by covalently closed circular structures, and they widely exist in mammalian cells. The aberrant expression of circRNAs may result in various diseases. Herein, we demonstrate the construction of genetically encoded light-up RNA aptamers for ultrasensitive and label-free detection of circRNA mitochondrial tRNA translation optimization 1 (circMTO1) in cancer cells and tissues. The lightup RNA aptamers are generated by proximity ligation-activated recombinase polymerase amplification (RPA)-assisted transcription amplification. When circMTO1 is present, it initiates the proximity ligation reaction, activating RPA to produce numerous long double-stranded DNAs containing T7 promoters. Subsequently, the RPA products are identified by T7 RNA polymerase, initiating the transcription amplification reaction to generate abundant Spinach RNA aptamers. Spinach RNA aptamers can bind with DFHBI (3,5-difluoro-4hydroxybenzylidene imidazolidinone) dye to produce a distinct fluorescence signal with near-zero background. This biosensor exhibits excellent selectivity and high sensitivity with a limit of detection of 2.54 aM. It can accurately monitor cellular circMTO1 at the single-cell level and discriminate the expression of circMTO1 between breast cancer patient tissues and healthy tissues. Notably, this biosensor can be employed to measure other nucleic acids by altering the corresponding target recognition sequences, providing a valuable platform for cancer diagnosis and biomedical study.

Microfluidic Chips Fabrication and Integration for Tumor‐Derived Exosomes Detection

“…successfully demonstrated that breast cancer patients have higher circMTO1 expression levels using a fluorescent detection strategy. [ 47 ] What is more, high expression of plasma exosome circPDE8A correlates with tumor progression and prognosis and would be used as a promising diagnostic or progression biomarker for Pancreatic ductal adenocarcinoma (PDAC). [ 48 ] However, the detection of circRNAs still faces great challenges.…”

Construction of Fluorescent G-Quadruplex Nanowires for Label-Free and Accurate Monitoring of Circular RNAs in Breast Cancer Cells and Tissues with Low Background

“…The qRT-PCR can readily quantify circRNA with relatively high sensitivity, but it involves reverse transcription that is susceptible to contamination. To overcome these drawbacks, a series of fluorescent and electrochemical methods integrated with nucleic acid amplification strategies (e.g., duplex-specific nuclease-mediated cycling amplification, , loop-mediated isothermal amplification, , catalytic hairpin assembly, − primer exchange reaction, hybridization chain reaction, and recombinase polymerase amplification) have been developed for circRNA assay with merits of improved sensitivity and more flexible sensing modes. However, these methods inevitably involve costly fluorophore/quencher dual-labeled signal probes, superabundant enzymes, and time-consuming operational procedures.…”