Allosteric Genetically Encoded Dual-Color Fluorogenic RNA Biosensor Simultaneously Illuminates miRNA and Its Precursor

Pre-miRNAs and miRNAs have garnered extensive attention due to their critical roles in various physiological and pathological processes. The intrinsic relationship between these biomarkers provides valuable insights into disease mechanisms and early diagnosis. Herein, we construct a dual-color fluorogenic RNA biosensor based on a hairpin allosteric-driven transcription circuit for simple, sensitive, and label-free monitoring of miRNA and pre-miRNA in cells and tissues. In this biosensor, we elaborated a multifunctional hairpin probe that integrates target recognition, transcription amplification, and signal transduction without additional auxiliary primers. The presence of miR-155 and pre-miR-155 can initiate their corresponding hairpin allosteric-driven transcription circuit to generate abundant Corn and Pepper aptamers, respectively, which subsequently bind with DFHO and HBC620 to transmit high fluorescence signals. By leveraging the high specificity and amplification efficiency of hairpin allosteric-driven transcription circuit and superior signal-to-noise ratio of RNA aptamer-fluorophore complexes, this biosensor can achieve isothermal, one-step, and homogeneous detection of miRNA and pre-miR-155 with attomolar sensitivity. Moreover, it enables the accurate quantification of miRNA and pre-miR-155 at the single-cell level and discrimination of their expression profiles between breast cancer and healthy tissues. Importantly, this biosensor can be scalable to explore other nucleic acids by altering the target binding sequence of a hairpin, providing a new paradigm for precise diagnosis.