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

作者：Zhao, Ning-ning; Li, Feng-zheng; Zhang, Xinyi; Liu, Meng; Cao, Hua^*; Zhang, Chun-yang^*

来源：Analytical Chemistry, 2023, 95(22): 8728-8734.

DOI：10.1021/acs.analchem.3c01624

• CIRCULAR RNA
• AMPLIFICATION
• CIRCMTO1

Circular RNAs (circRNAs) as endogenous non-coding RNAsare characterizedby covalently closed circular structures, and they widely exist inmammalian cells. The aberrant expression of circRNAs may result invarious diseases. Herein, we demonstrate the construction of geneticallyencoded light-up RNA aptamers for ultrasensitive and label-free detectionof circRNA mitochondrial tRNA translation optimization 1 (circMTO1)in cancer cells and tissues. The light-up RNA aptamers are generatedby proximity ligation-activated recombinase polymerase amplification(RPA)-assisted transcription amplification. When circMTO1 is present,it initiates the proximity ligation reaction, activating RPA to producenumerous long double-stranded DNAs containing T7 promoters. Subsequently,the RPA products are identified by T7 RNA polymerase, initiating thetranscription amplification reaction to generate abundant SpinachRNA aptamers. Spinach RNA aptamers can bind with DFHBI (3,5-difluoro-4-hydroxybenzylideneimidazolidinone) dye to produce a distinct fluorescence signal withnear-zero background. This biosensor exhibits excellent selectivityand high sensitivity with a limit of detection of 2.54 aM. It canaccurately monitor cellular circMTO1 at the single-cell level anddiscriminate the expression of circMTO1 between breast cancer patienttissues and healthy tissues. Notably, this biosensor can be employedto measure other nucleic acids by altering the corresponding targetrecognition sequences, providing a valuable platform for cancer diagnosisand biomedical study.

• 单位
  广东药学院