Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag

Abstract

Nanopores, both protein and solid-state, are explored as single molecule analytical tools, but using an experimental platform is challenging. Here we show that a commercially available nanopore device, MinION from Oxford Nanopore Technologies (ONT), successfully accomplishes a task challenging for a conventional analytical tool. Specifically the MinION discriminates among 31 nucleotide (nt) long oligoriboadenylates with a single pyrimidine (Py) substitution, when this pyrimidine is tagged/labeled with a bulky group (Osmium tetroxide 2,2’-bipyridine or OsBp). This platform also discriminates between an osmylated Py (Py-OsBp) followed by a purine (Pu) and a Py-OsBp followed by a second Py-OsBp, leading to the conjecture that the bulky tag enables sensing of a two-nucleotide sequence. Two-nucleotide sensing could greatly improve base-calling accuracy in motor enzyme-assisted nanopore sequencing.We attribute the observed discrimination neither to the specific pore protein nor to OsBp, but to the tag’s bulkiness, that leads to markedly slower translocation and “touching” proximity at the pore’s constriction zone, that forces desolvation and reorganization, and enables strong interactions among the nanopore, the tagged pyrimidine, and the adjacent nucleobase. These results constitute proof-of-principle that size-suitable nanopores may be superior to traditional analytical tools, for the characterization of RNA oligos and microRNAs enhanced by selective labelling.