Identification of nucleoside monophosphates and their epigenetic modifications using an engineered nanopore

RNA modifications play critical roles in the regulation of various biological processes and are associated with many human diseases. Direct identification of RNA modifications by sequencing remains challenging, however. Nanopore sequencing is promising, but the current strategy is complicated by sequence decoding. Sequential nanopore identification of enzymatically cleaved nucleoside monophosphates may simultaneously provide accurate sequence and modification information. Here we show a phenylboronic acid-modified hetero-octameric Mycobacterium smegmatis porin A nanopore, with which direct distinguishing between monophosphates of canonical nucleosides, 5-methylcytidine, N⁶-methyladenosine, N⁷-methylguanosine, N¹-methyladenosine, inosine, pseudouridine and dihydrouridine was achieved. A custom machine learning algorithm, which reports an accuracy of 0.996, was also applied to the quantitative analysis of modifications in microRNA and natural transfer RNA. It is generally suitable for sensing of a variety of other nucleoside or nucleotide derivatives and may bring new insights to epigenetic RNA sequencing.