Multiplexed miRNA detection based on target-triggered transcription of multicolor fluorogenic RNA aptamers

Abstract

We herein describe a new multicolor fluorogenic RNA aptasensor to accomplish multiplexed detection of miRNAs. The stem-loop primer (SL primer) entailing a fluorogenic RNA aptamer (FRA) antisense sequence is designed to anneal to target miRNA at its 3' overhang, which would be reverse transcribed by reverse transcriptase (RT) to produce the cDNA sequence followed by the degradation of target miRNA. The T7 promoter-containing primer (T7 primer) is then annealed to the 3' end of the extended cDNA sequence and the following RT-promoted extension in both directions produces the T7 promoter-containing double-stranded DNA (T7 dsDNA). T7 RNA polymerase finally transcribes the T7 dsDNA to produce a large number of RNA transcripts containing FRA sequence, which would produce intense fluorescence signals by forming fluorescent complexes with cognate fluorogens, reflecting the amount of target miRNAs. Based on this unique design principle employing the SL primers to encode several different FRAs with distinct fluorescence profiles, target miRNAs were very specifically determined in a multiplexed manner down to a subpicomolar level. The practical applicability of this technique was also verified by reliably quantifying target miRNAs in serum and human cancer cell lysates.

Keywords: Fluorogenic RNA aptamer; In vitro transcription; MicroRNA detection; Stem-loop primer.