Direct microRNA sequencing using Nanopore Induced Phase-Shift Sequencing (NIPSS)

Abstract

MicroRNAs (miRNAs) are a class of short non-coding RNAs that function in RNA silencing and post-transcriptional gene regulation. Besides their participation in regulating normal physiological activities, specific miRNA types could act as oncogenes, tumor suppressors or metastasis regulators, which are critical biomarkers for cancer. However, direct characterization of miRNA is challenging due to its unique properties such as its low abundance, sequence similarities and short length. 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 demonstrates the first report of direct miRNA sequencing, which serves as a complement to existing miRNA sensing routines by the introduction of single molecule resolution. Future engineering of this technique may assist miRNA based early stage diagnosis or inspire novel cancer therapeutics.MicroRNAs (miRNAs) are a group of short, single-stranded, non-coding RNA molecules that act as posttranscriptional gene regulators for a wide variety of physiological processes, including proliferation, differentiation, apoptosis, and immune reactions 1, 2 . On the other hand, aberrant miRNAs expression levels have been shown to be closely related to diverse diseases, such as cancer 3-6 , auto-immune disorders 7 and inflammatory diseases 8 .Conventionally, miRNAs can be characterized by northern blot, quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) assays or microarrays 9 .Other emerging platforms for miRNA sensing include colorimetry 10 , bioluminescence 11 , enzymatic activity 12 , nanopore sensing 13 and electrochemistry 14 . Unfortunately, these methods provide limited analytical information because miRNA sequences are not directly reported and prior knowledge of the target miRNA sequence is required. MiRNAs function by binding to the 3' untranslated region (3'UTR) of target messengerRNAs 15 . Minor sequence variations, which include trimming, addition or substitution of miRNA sequences, alter its binding affinities to target messenger RNA 15 . As reported, miRNA isoforms (isomiRs) 16 , which were generated by the addition or deletion of one or multiple nucleotides as terminal modifications, have been shown to participate in proliferative diseases 17 and cancer 18 . On the other hand, N6-methyl-adenosine (m6A), which is an epigenetic modification among RNAs, plays important roles in miRNA biogenesis 19, 20 . These functional roles of miRNAs are associated with their specific sequences. Therefore, directly decoding a miRNA sequence along with its chemical modifications becomes critical to correlate its physiological or pathological relevance.MiRNA could be indirectly sequenced by sequencing its complementary DNAs (cDNA) by performing reverse transcription followed with deep sequencing 21 . However, this strategy suffers f...