Sequencing for RNA modifications with the nanopore direct RNA sequencing platform provides ionic current levels, helicase dwell times, and base call data that differentiate the modifications from the canonical form. Herein, model RNAs were synthesized with site-specific uridine (U) base modifications that enable the study of increasing an alkyl group size, halogen identity, or a change in base acidity to impact the nanopore data. The analysis concluded that increases in alkyl size trend with greater current blockage but a similar change in base-call error was not found. The addition of a halogen series to C5 of U revealed that the current levels recorded a trend with the water-octanol partition coefficient of the base, as well as the base call error. Studies with U modifications that are deprotonated (i.e., anionic) under the sequencing conditions gave broad current levels that influenced the base call error. Some modifications led to helicase dwell time changes. These insights provide design parameters for modification-specific chemical reagents that can shift nanopore signatures to minimize false positive reads, a known issue with this sequencing approach.

Keywords: 5-bromouridine; 5-ethynyluridine; 5-hydroxyuridine; Nanopore sequencing; RNA modifications; pseudouridine.