Development of a Label-Free Fluorescence Strategy for Sensitive and Site-Specific Measurement of RNA Inosine...

Inosine (I) is an inert purine nucleoside that exerts critical regulatory effects on RNA maturation, alternative splicing, and translational fidelity. Dysregulation of inosine modifications has been tightly associated with the pathogenesis of diverse human diseases including neurological conditions, autoimmune diseases, and tumorigenesis. Herein, we propose a label-free fluorescence strategy based on cyanoethylation-induced elongation arrest and an exonuclease I-catalyzed digestion reaction for sensitive and site-specific detection of RNA inosine modification in human cell lines and breast tissues. In this assay, target inosine in RNA (I-RNA) can be selectively cyanoethylated with acrylonitrile to form an N¹-cyanoethylinosine-modified RNA (ce¹I-RNA) via Michael addition reaction, followed by the hybridization with the bifunctional probe modified on the magnetic bead surface to form a DNA/RNA heteroduplex. The resultant ce¹I can block DNA polymerase-mediated reverse transcription, and exonuclease I can digest the excessive free bifunctional probes on the magnetic bead surface. Consequently, the remaining bifunctional probes can initiate the primer generation-rolling circle amplification (PG-RCA) to produce numerous single-stranded DNA products that may be easily measured with SYBR Gold as the stainer in a label-free manner. This strategy possesses excellent specificity, high sensitivity, and a low background. It can achieve an ultralow detection limit of 67.97 aM and enables the discrimination of as low as 0.01% inosine in mixed nucleotide samples and accurate quantification of RNA inosine at the single-cell level. Importantly, this method can successfully distinguish inosine expressions between the clinical breast cancer tissues and their normal counterparts, with potential applications in inosine-associated biological studies and clinical diagnosis.