RESUMEN
Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.
RESUMEN
Photobases are a type of molecule that become more basic upon photoexcitation and can therefore be used to control proton transfer reactions with light. The solvation requirements for excited state proton transfer (ESPT) in photobase systems is poorly understood, which limits their applicability. Here, we investigate the solvation of the ESPT reaction using 5-methoxyquinoline (MeOQ), a well-studied photobase with an excited state pKa (pKa*) of approximately 15.1, as a model system. Previous studies have shown that, in addition to the acidic donor that donates a proton to the photoexcited MeOQ, an additional "auxiliary donor" is necessary to solvate the resulting alkoxide. We investigate whether a nonacidic hydrogen bond donor (an alcohol solvent that MeOQ cannot deprotonate in bulk) can act as the auxiliary donor for the MeOQ ESPT reaction. First, we use steady state spectroscopy, TCSPC, and electronic structure calculations to show that MeOQ can deprotonate the acidic donor 2,2,2-trifluoroethanol (TFE, pKa = 12.5) using ethanol as the auxiliary donor. We show that the degree of ESPT is largely predicted by the degree of ground state hydrogen bonding between the photobase and the acidic donor. Next, we study the deprotonation of the acidic donors TFE and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, pKa = 9.3) with MeOQ in a variety of nonacidic alcohol solvents of varying chain length and branching. MeOQ ESPT occurs to varying extents in all solvents, suggesting that all studied nonacidic alcohols can function as auxiliary donors. We show that the concentration of the acidic donor is strongly correlated with the degree of ESPT. These results are necessary fundamental steps toward the understanding of the photobase ESPT reaction and its wide application in a variety of chemical systems.