Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond

Abstract

RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.

Keywords: RNA; SHAPE; capillary electrophoresis; chemical probe; enzymatic probe; high-throughput sequencing; mutational profiling; structure.

Figure 1

Enzymatic and chemical probes available nowadays. (A) Common enzymatic probes and their targets on ss- and dsRNA. The arrows and the highlighted nucleotides indicate whether the fragment formed after strand scission is 3′ or 5′ phosphate. (B) Main chemical probes and their target positions on base, sugar, and phosphate. DMS: dimethylsulfate; CMCT: 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DEPC: diethylpyrocarbonate; kethoxal: 3-ethoxy-1,1-dihydroxy-2-butanone; SHAPE reagents are NMIA: N-methylisatoic anhydride, BzCN: benzoylcyanide, and NAI: 2-methyl nicotinic acid imidazolide; FAI: 2-methyl-3-furoic acid imidazolide; 1M6: 1-methyl-6-nitroisatoic anhydride; 1M7: 1-methyl-7-nitroisatoic anhydride and 2A3: 2-aminopyridine-3-carboxylic acid imidazolide; NAz: nicotinoyl-azide; Glyoxal: ethanedial [40]; ENU: ethyl-nitrosourea; Pb(II): lead ion; ^•OH: hydroxyl radical.

Figure 2

Timeline of RNA probes and probing techniques evolution. Upper part, enzymes are boxed in blue, chemical reagents (with the exception of SHAPE reagents) are boxed in orange, and SHAPE probes are boxed in green; the center of the box corresponds to the year of publication. Lower part, the blue arrows indicate the implementation of a new method to detect sites of cleavage or modification. CE: capillary electrophoresis; Seq: deep-sequencing; MaP: mutational profiling. Methods combining probing and deep-sequencing are highlighted in yellow. Methods based on mutational profiling are highlighted in orange, and techniques using enrichment by selection via the probe are labeled in purple. PARS: parallel analysis of RNA structure; FragSeq: fragmentation sequencing; CIRS-Seq: DMS and CMCT probing and sequencing; HRF-Seq: hydroxyl radical cleavage and sequencing; Mod-Seq: DMS probing and sequencing; Structure-Seq: DMS probing and sequencing; SHAPES: probing with NPIA and selection; icSHAPE: probing with NAI-N₃ and selection; Keth-seq: N₃-kethoxal probing, selection, and sequencing; LASER/-seq/-MaP: light-activated structural examination of RNA/analyzed by sequencing/mutational profiling.

Figure 3

Schematic representation of RNA probing methods based on chemical modification. Folded RNAs were modified with the chemical probe of interest (green panel). For the RT stop strategy (left, blue panel), modified RNAs were reverse-transcribed with (1) radiolabeled primers for gel electrophoresis visualization (left, yellow panel), (2) fluorescently labeled primers for capillary electrophoresis detection (center, yellow panel), or (3) unlabeled primers for high-throughput sequencing after adapter ligation and library preparation (right, yellow panel). For the mutational profiling approach (right, blue panel), the modified RNAs were reverse-transcribed under mutation-inducing conditions with unlabeled primers. The cDNAs were ligated to adapters, and a library was prepared for high-throughput sequencing (right, yellow panel). The main advantages and disadvantages of the three analysis methods were featured at the bottom of the yellow panel.