Advances in RNA structure analysis by chemical probing

Abstract

RNA is arguably the most versatile biological macromolecule because of its ability both to encode and to manipulate genetic information. The diverse roles of RNA depend on its ability to fold back on itself to form biologically functional structures that bind small molecule and large protein ligands, to change conformation, and to affect the cellular regulatory state. These features of RNA biology can be structurally interrogated using chemical mapping experiments. The usefulness and applications of RNA chemical probing technologies have expanded dramatically over the past five years because of several critical advances. These innovations include new sequence-independent RNA chemistries, algorithmic tools for high-throughput analysis of complex data sets composed of thousands of measurements, new approaches for interpreting chemical probing data for both secondary and tertiary structure prediction, facile methods for following time-dependent processes, and the willingness of individual research groups to tackle increasingly bold problems in RNA structural biology.

Figure 1

Classes of RNA structure information obtained by chemical probing include (A) base selective data, (B) solvent accessibility information, (C) measurements of nucleotide dynamics, and (D) constraints on long-range interactions.

Figure 2

Sites of RNA modification for base-selective (left) and sequence-independent (right) modification chemistries. DEPC and CMCT also react with guanosine (not shown).

Figure 3

Automated analysis of chemical probing experiments using the (A) SAFA and (B) ShapeFinder programs. Figures adapted from [18,56].

Figure 4

Comparison of secondary structure prediction accuracy in the 5′ domain of E. coli 16S rRNA using free energy minimization (A) alone, (B) with conventional chemical modification reagents, and (C) with SHAPE-derived pseudo-free energy change terms. Missed base pairs are indicated by red x’s; incorrectly predicted base pairs are represented by colored lines. Regions where experimental information supports local RNA refolding are indicated with green boxes and spheres in panel C. Figure adapted from [27].

Figure 5

Recent developments using chemical probing to obtain through-space constraints on RNA tertiary structure. (A) MOHCA, (B) MS3D, and (C) sequence-encoded cleavage. Figures are adapted from images in [46,48,49].

Figure 6

Representative examples of the use of chemical probing technologies to address ambitious problems in biology. Adapted from [31,34,55].