Highlights

• Transfer RNAs (tRNAs) are identified as the most heavily modified RNA species, setting the stage for novel research in RNA modification analysis.
• A groundbreaking approach involving the hydrolysis of target RNA to nucleosides using nonspecific ribonuclease (RNase I), followed by LC-MS analysis based on retention time (Rt) and mass-to-charge ratio (m/z), has been developed. This method identifies modified nucleosides with extreme sensitivity, capturing attomole quantities.
• Pioneering work on the chimeric RNA/DNA-RNase H approach for full-range profiling of tRNA modifications on an LC-MS platform has been introduced. Additionally, the study presents innovative findings regarding the simultaneous recording of ionized nucleotide and nucleobase signals through direct collision-induced dissociation of source oligonucleotides, further advancing the field of RNA analysis.

Summary

A novel method has been developed to comprehensively profile modifications in transfer RNAs (tRNAs) at a single-base resolution. This method combines RNase H digestion, LC-MS/MS, and RNA-seq techniques, enabling the sequencing of post-transcriptionally modified tRNAs with high sequence coverage.

In ‘Full-Range Profiling of tRNA Modifications Using LC–MS/MS at Single-Base Resolution through a Site-Specific Cleavage Strategy’, Tingting Yan et al. (2020) reported that transfer RNAs are heavily modified RNA species. A chimeric RNA/DNA-RNase H approach was used to analyze tRNA modifications. The cleavage in the stem regions of tRNAs yielded better results than in loops. False positives were reduced by 95% using new inclusion criteria. A novel approach based on site-specific digestion was used to collect accurate census data.

Transfer RNAs are RNA molecules that undergo extensive modifications, and LC-MS/MS is employed to identify these modified nucleosides based on their retention time and mass-to-charge ratio. Utilizing highly sensitive LC-MS methods, even minute quantities of nucleosides can be captured.

A hybrid RNA/DNA-RNase H approach is utilized to profile tRNA modifications, focusing on cleaving the stem regions of tRNAs to yield better results. Automated research algorithms are employed to eliminate false positives during data analysis.

The identified modifications are subsequently mapped back into the gene sequence of the respective tRNA molecules. Specifically, pseudouridine modifications can be identified through cyanoethylation.

Notably, LC-MS/MS data from PART-4 could not be analyzed in the fixed mode.

Overall, this systematic method combining RNase H digestion, LC-MS/MS, and tRNA-seq techniques allows for the profiling and characterizing modifications in various tRNA species, including mouse mitochondrial tRNAHis(GUG) and tRNAVal(UAC). This method offers a robust pathway for sequencing heavily modified post-transcriptional tRNAs with high sequence coverage.

It’s also worth mentioning that some of the research findings in this study differ from previous research in the field. The researchers highlight using the powerful software tool “RAMM” to eliminate false positives and accurately interpret the MS/MS spectra of tRNA digestion products. However, it’s noted that RAMM did not yield correct interpretations for the heavily modified anticodon regions in the variable mode, as mentioned by Yan.

For further details and access to the data and code related to this research, you can refer to the following link: https://pubs.acs.org/doi/10.1021/acs.analchem.0c03307.

H.-X. Zhang et al., “Selective Chemical Labeling Strategy for Oligonucleotides Determination: A First Application to Full-Range Profiling of Transfer RNA Modifications,” Anal. Chem., p. acs.analchem.2c02302, Jan. 2023, doi: .