MicroRNAs (miRNAs) are a class of short non-coding RNAs that function in RNA silencing and post-transcriptional gene regulation. However, direct characterization of miRNA is challenging due to its unique properties such as its low abundance, sequence similarities, and short length. Although urgently needed, single molecule sequencing of miRNA has never been demonstrated, to the best of our knowledge. Nanopore-induced phase-shift sequencing (NIPSS), which is a variant form of nanopore sequencing, could directly sequence any short analytes including miRNA. In practice, NIPSS clearly discriminates between different identities, isoforms, and epigenetic variants of model miRNA sequences. This work thus demonstrates direct sequencing of miRNA, which serves as a complement to existing miRNA sensing routines by the introduction of the single molecule resolution. Future engineering of this technique may assist miRNA-based early stage diagnosis or inspire novel cancer therapeutics.
Direct miRNA Sequencing Using NIPSS
(A) A schematic diagram of the preparation of a sequencing library. The miRNA sequencing library is thermally annealed from three separate nucleic acid strands, which include a chimeric template, a primer (green), and a blocker (light blue). The chimeric template is composed of a miRNA segment (red), an abasic residue (blue dot), and a DNA segment (black). (B) The NIPSS strategy for direct miRNA sequencing. NIPSS is carried out with an MspA nanopore (purple) and a wild-type (WT) phi29 DNAP (green) by following the reported enzymatic ratcheting strategy. (C) A typical current trace acquired by sequencing DNA-miR-21 using NIPSS. (D) Overlay of multiple time-normalized events (N = 24) from the DNA-miR-21 results acquired by NIPSS.