An ultrasensitive label-free RNase H assay based on in vitro transcription of fluorogenic light-up aptamer

Abstract

Herein, we proposed a label-free method to identify RNase H activity by utilizing in vitro transcription of fluorogenic light-up aptamers. In this work, we employed the specially designed two pivotal components of the hairpin substrate probe (HP) containing an RNA/DNA chimeric stem region and the template probe (TP) as a transcription template, and the RNase H activity was made to lead to the formation of a complete ds T7 promoter. T7 RNA polymerase could then promote in vitro transcription to generate numerous light-up RNA aptamers that result in significant fluorescence enhancements upon binding to the cognate fluorogenic dye. By leveraging this deliberate design principle, we identified RNase H activity ultrasensitively as low as 0.000156 U mL⁻¹ with excellent specificity against non-target enzymes. We further demonstrated that the strategy can also reliably identify RNase H activity in heterogeneous biological samples such as cell lysates, ensuring its robust practical applicability. This work would provide invaluable insight for the development of innovative biosensing systems utilizing in vitro transcription of light-up aptamers, and it could be broadened to construct other assays by appropriately redesigning the HPs.