Analysis of RNA and Its Modifications

Abstract

Ribonucleic acids (RNAs) are key biomolecules responsible for the transmission of genetic information, the synthesis of proteins, and modulation of many biochemical processes. They are also often the key components of viruses. Synthetic RNAs or oligoribonucleotides are becoming more widely used as therapeutics. In many cases, RNAs will be chemically modified, either naturally via enzymatic systems within a cell or intentionally during their synthesis. Analytical methods to detect, sequence, identify, and quantify RNA and its modifications have demands that far exceed requirements found in the DNA realm. Two complementary platforms have demonstrated their value and utility for the characterization of RNA and its modifications: mass spectrometry and next-generation sequencing. This review highlights recent advances in both platforms, examines their relative strengths and weaknesses, and explores some alternative approaches that lie at the horizon.

Keywords: LC-MS/MS; RNA sequencing; epitranscriptome; modified nucleosides; post-transcriptional modifications; therapeutic RNAs.

Figure 1.. Approaches for Analyzing RNA and its Modifications.

Overview of most common approaches for analyzing oligoribonucleotides and RNA sequences including those that are modified, either biologically or for therapeutic applications. Abbreviations: LC-MS/MS, liquid chromatography tandem mass spectrometry; RNA-Seq, RNA sequencing (next generation sequencing).

Figure 2.. Capturing dynamic changes in RNA modifications.

High resolution mass spectra of stable isotope labeled nucleosides from cell culture. (a) Labeling of compounds used for stable isotope labeling in cell culture. Grey circles indicate the positions of isotopes (¹³C, ¹⁵N, or ²H/D). (b) Merged high resolution mass spectra of the four canonical nucleosides of total tRNA after labeling of HEK 293 cells with shown compounds for 7 days. Background signals are marked with asterisks. (c) Merged high resolution mass spectra of three exemplary modifications (m⁵C, m⁷G, and m¹A) in total tRNA after stable isotope labeling of HEK 293 cells for 7 days. Figure adapted with permission from Reference (14); copyright 2021 The Authors. Abbreviations: tRNA, transfer ribonucleic acid; HEK 293, Human embryonic kidney 293; m⁵C, 5-methylcytidine; m⁷G, 7-methylguanosine; m¹A, 1-methyladenosine.

Figure 3.. Automating the detection of modified nucleosides by mass spectral networks.

Graph depicting a spectral network of ribonucleosides. For easier identification, canonical ribonucleosides (cytidine, uridine, guanine, and adenosine) are represented by larger nodes. Examples of clusters formed by structurally related modified nucleosides, as well as their shared structural cores and MS/MS spectra, are highlighted on inset A, inset B, and inset C. An interactive version of the spectral network may be found at http://bearcatms.uc.edu/spectral-network-interactive/. Figure adapted with permission from Reference (25); copyright 2022, American Chemical Society. Abbreviations: MS/MS, tandem mass spectrometry.

Figure 4.. Enhancing the separation of oligonucleotides.

Separation of 15, 20 25, 30, 35, 40, 50, and 60 mer oligodeoxythymidines using selected 100 mM ion-pairing buffers. The data are compared to non-ion-pairing 25 mM ammonium acetate buffer. Longer alkyl-chain ion-pairing buffers lead to enhanced separation of oligonucleotides. Figure adapted with permission from Reference (32); copyright 2022 Elsevier. Abbreviations: AA, ammonium acetate; DEAA, diethylammonium acetate; TEAA, triethylammonium acetate; BAA, butylammonium acetate; DBAA, dibutylammonium acetate; OAA, octylammonium acetate.

Figure 5.. The use of native mass spectrometry to understand RNA binding.

Binding site mapping of RRE-TR- 0/rev complexes by CAD MS. (a) Site-specific occupancies (O) of c (left axis) and y (right axis) fragments with rev ARM peptide from CAD of 1:1 complex ions, (RRE-TR-0+ 1·rev - 14H)14−, at 137.2 eV and the corresponding binding region (blue) mapped onto the predicted secondary structure of RRE-TR-0 (b) show poor agreement with binding sites in the NMR structure (e). (c) Occupancies of fragments from CAD of 1:2 complex ions, (RRE-TR-0+ 2·rev - 14H)14−, at 175.5 eV and corresponding binding sites (violet) mapped onto the RRE-TR-0 structure (d) show good agreement with binding sites in the NMR structure (e). Darker and lighter colors in b, d, and e stand for stronger and weaker binding, respectively. Panel adapted with permission from Reference (91); copyright 2020 Nature Communications. Abbreviations: CAD, collision activated disassociation mass spectrometry; ARM, arginine-rich motif; NMR, nuclear magnetic resonance.

Figure 6.. Enhanced detection of m⁶A sites in RNA.

Sanger sequencing traces showing C-to-U editing adjacent to m⁶A sites in cells expressing APO1-YTHD422N, APO1-YTH, and APO1-YTHmut for five mRNAs previously shown to contain m⁶A: DPM2, EIF4B, HERC2, NIPA1, and SMUG1. m⁶A sites are indicated by asterisks. C-to-U editing rate (%U) is indicated above the adjacent cytidine. Data are representative of three biological replicates. Panel adapted with permission from Reference (98) copyright 2022; Frontiers in Cell and Developmental Biology. Abbreviations: mRNA, messenger RNA.