ABSTRACT
Capturing the folding dynamics of large, functionally important RNAs has relied primarily on global measurements of structure or on per-nucleotide chemical probing. These approaches infer, but do not directly measure, through-space structural interactions. Here we introduce trimethyloxonium (TMO) as a chemical probe for RNA. TMO alkylates RNA at high levels in seconds, and thereby enables time-resolved, single-molecule, through-space probing of RNA folding using the RING-MaP correlated chemical probing framework. Time-resolved correlations in the RNase P RNA-a functional RNA with a complex structure stabilized by multiple noncanonical interactions-revealed that a long-range tertiary interaction guides native RNA folding for both secondary and tertiary structure. This unanticipated nonhierarchical folding mechanism was directly validated by examining the consequences of concise disruption of the through-space interaction. Single-molecule, time-resolved RNA structure probing with TMO is poised to reveal a wide range of dynamic RNA folding processes and principles of RNA folding.
Subject(s)
Onium Compounds/chemistry , RNA/chemistry , Alkylation , Base Pairing , Nucleic Acid Conformation , RNA/metabolism , RNA Folding , Ribonuclease P/metabolism , Sulfuric Acid Esters/chemistryABSTRACT
RNA viruses encode the information required to usurp cellular metabolism and gene regulation and to enable their own replication in two ways: in the linear sequence of their RNA genomes and in higher-order structures that form when the genomic RNA strand folds back on itself. Application of high-resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) structure probing to viral RNA genomes has identified numerous new regulatory elements, defined new principles by which viral RNAs interact with the cellular host and evade host immune responses, and revealed relationships between virus evolution and RNA structure. This review summarizes our current understanding of genome structure-function interrelationships for RNA viruses, as informed by SHAPE structure probing, and outlines opportunities for future studies.
