Direct RNA sequencing for chemical modifications is possible with single-molecule nanopore sequencing. Sequencing signatures in the form of ionic current versus time traces are recorded as the RNA is electrophoretically driven through a small aperture nanopore in which a helicase enzyme slowly ratchets the RNA through the nanopore enabling data recording. Nucleotide chemical modifications are found by inspecting the data for base-calling errors, or ionic current and dwell-time differences, in comparison to the sequencing of control RNA without the modifications. Ribosomal RNA from natively grown E. coli cells that possess 36 chemical modifications of 17 different types were directly sequenced with the nanopore to identify unique base-calling errors, ionic current, and/or dwell-time signatures for many of the modification types. The data were analyzed to provide semi-quantitation of the modifications. The E. coli cells were then subjected to stressors followed by resequencing the rRNA to identify decreases in the levels of target base and sugar modifications. Specific pseudouridine and N2-methylguanosine residues in the 16S and 23S rRNAs were found to decrease. The helicase dwell-time data permitted us to identify the single methyl group lost from N4-methyl-2`-O-methylcytidine in the 16S rRNA during thermal stress. Loss of a single methyl group at this cytidine can trigger hibernation. These results are a major step toward the Holy Grail of single-molecule sequencing for simultaneous identification and quantitation of all nucleotide chemical modifications.

Nanopore dwell-time analysis identifies stress-dependent rRNA modifications

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.