2d-hels-ms-seq-general-lc-ms-based-method-for-direct-de-novo

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

New York Institute of Technology

2΄-5΄-linked RNAs play important roles in many biological systems. In addition, the mixture of 2΄-5΄ and 3΄-5΄ phosphodiester bonds have emerged as a plausible structural element in prebiotic RNAs. Toward our mechanistic studies of RNA folding and structures with heterogeneous backbones, we recently reported two crystal structures of a decamer RNA duplex containing two and six 2΄-5΄-linkages, showing how RNA duplexes adjust the structures to accommodate these non-canonical linkages (Proc. Natl. Acad. Sci. USA, 2014, 111, 3050-3055). Herein, we present two additional high-resolution crystal structures of the same RNA duplex containing four and eight 2΄-5΄-linkages at different positions, providing new insights into the effects of these modifications and a dynamic view of RNA structure changes with increased numbers of 2΄-5΄-linkages in the same duplex. Our results show that the local structural perturbations caused by 2΄-5΄ linkages can be distributed to nearly all the nucleotides with big ranges of changes in different geometry parameters. In addition, hydration pattern and solvation energy analysis indicate less favorable solvent interactions of 2΄-5΄-linkages comparing to the native 3΄-5΄-linkages. This study not only promotes our understanding of RNA backbone flexibility, but also provides a knowledge base for studying the biochemical and prebiotic significance of RNA 2΄-5΄-linkages.

Structural insights into RNA duplexes with multiple 2΄-5΄-linkages.

A complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and non-canonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these non-standard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via introduction of a 2-dimensional hydrophobic end-labeling strategy into traditional mass spectrometry-based sequencing (2D HELS MS Seq) to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA versus its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site in the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification type and can also be used in the quality control of modified therapeutic RNAs.

A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures.

Post-transcriptional modifications are intrinsic to RNA structure and function. However, methods to sequence RNA typically require a cDNA intermediate and are either not able to sequence these modifications or are tailored to sequence one specific nucleotide modification only. Interestingly, some of these modifications occur with <100% frequency at their particular sites, and site-specific quantification of their stoichiometries is another challenge. Here, we report a direct method for sequencing tRNA without cDNA by integrating a two-dimensional hydrophobic RNA end-labeling strategy with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNA was sequenced and the identity, location, and stoichiometry of all eleven different RNA modifications was determined, five of which were not 100% modified, including a 2'-O-methylated G (Gm) in the wobble anticodon position as well as an -dimethylguanosine (mG), a 7-methylguanosine (mG), a 1-methyladenosine (mA), and a wybutosine (Y), suggesting numerous post-transcriptional regulations in tRNA. Two truncated isoforms at the 3'-CCA tail of the tRNA (75 nt with a 3'-CC tail (80% abundance) and 74 nt with a 3'-C tail (3% abundance)) were identified in addition to the full-length 3'-CCA-tailed tRNA (76 nt, 17% abundance). We discovered a new isoform with A-G transitions/editing at the 44 and 45 positions in the tRNA variable loop, and discuss possible mechanisms related to the emergence and functions of the isoforms with these base transitions or editing. Our method revealed new isoforms, base modifications, and RNA editing as well as their stoichiometries in the tRNA that cannot be determined by current cDNA-based methods, opening new opportunities in the field of epitranscriptomics.

Article	Year
Structural insights into RNA duplexes with multiple 2΄-5΄-linkages. Nucleic acids research\| PubMed ID: 28034958	2017
A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures. Nucleic acids research\| PubMed ID: 31504795	2019
Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications. ACS chemical biology\| PubMed ID: 32364699	2020

Shenglong Zhang

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