High-sensitive sensing of plant microRNA by integrating click chemistry with an unusual on-bead poly(T)-promoted transcription amplification

Abstract MicroRNAs (miRNAs) act as pivotal regulators in plants. Therefore, sensing strategies with high specificity and high sensitivity are desired for plant miRNA analysis in order to unveil the exact biofunctions of miRNAs. Toward this goal, a fluorescent assay is developed based on a two-step signal amplification strategy. In the first step, target miRNA-templated cycling click nucleic acid ligation is employed for target recognition and amplification, the product of which can bind to magnetic microbeads (MBs) and introduce the T7 promoter sequence to the surface. In the second step, the poly(T) containing transcription template partially hybridizes with the T7 promoter sequence on the ligated strand and then regulates the on-bead transcription in a cycling manner with the participation of T7 RNA polymerase. Surprisingly, different from other reported templates, the poly(T) template improves the transcription efficiency to an unexpectedly high level by releasing ultra-long RNA chains in the reaction system. Finally, the RNA intercalating dye, RiboGreen, is utilized to specifically light up the as-produced RNA chains for low-background signal readout. Benefiting from the elaborate design, the detection limit of plant miRNA is down to ∼0.1 amol. This strategy provides a highly specific and highly sensitive platform for plant miRNA detection, which promises potential in the practical applications of miRNA-related biofunctions.