Synergistic SHAPE/Single-Molecule Deconvolution of RNA Conformation under Physiological Conditions

Abstract

Structural RNA domains are widely involved in the regulation of biological functions, such as gene expression, gene modification, and gene repair. Activity of these dynamic regions depends sensitively on the global fold of the RNA, in particular, on the binding affinity of individual conformations to effector molecules in solution. Consequently, both the 1) structure and 2) conformational dynamics of noncoding RNAs prove to be essential in understanding the coupling that results in biological function. Toward this end, we recently reported observation of three conformational states in the metal-induced folding pathway of the tRNA-like structure domain of Brome Mosaic Virus, via single-molecule fluorescence resonance energy transfer studies. We report herein selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)-directed structure predictions as a function of metal ion concentrations ([Mⁿ⁺]) to confirm the three-state folding model, as well as test 2° structure models from the literature. Specifically, SHAPE reactivity data mapped onto literature models agrees well with the secondary structures observed at 0-10 mM [Mg²⁺], with only minor discrepancies in the E hairpin domain at low [Mg²⁺]. SHAPE probing and SHAPE-directed structure predictions further confirm the stepwise unfolding pathway previously observed in our single-molecule studies. Of special relevance, this means that reduction in the metal-ion concentration unfolds the 3' pseudoknot interaction before unfolding the long-range stem interaction. This work highlights the synergistic power of combining 1) single-molecule Förster resonance energy transfer and 2) SHAPE-directed structure-probing studies for detailed analysis of multiple RNA conformational states. In particular, single-molecule guided deconvolution of the SHAPE reactivities permits 2° structure predictions of isolated RNA conformations, thereby substantially improving on traditional limitations associated with current structure prediction algorithms.

Figure 1

Stepwise folding dynamics in the TLS of BMV. The F conformation at high salt is stabilized by two intermolecular interactions—a stem (green) and a PSK (red). Decrease in [Mⁿ⁺] first breaks the 3′ PSK interaction before breaking the long-range stem. To see this figure in color, go online.

Figure 2

SHAPE assay. Outline of the four main steps in the SHAPE assay: (1) incorporation of the SHAPE structure cassettes, (2) SHAPE chemistry in 2′O-adduct formation, (3) fragmentation and cDNA formation in primer extension, and (4) analysis of fragmentation patterns via capillary electrophoresis. To see this figure in color, go online.

Figure 3

Single-molecule burst fluorescence. (a) Single-molecule probability distributions are shown of FRET efficiencies at 0–1000 mM [K⁺]. (b) Fractional distributions are shown of single-molecule burst studies on [Mⁿ⁺]-induced TLS folding experiments. TLS constructs in U conformation are shown in blue, I conformation in green, and F conformation in red. To see this figure in color, go online.

Figure 4

[Mⁿ⁺]-dependent SHAPE probing of the TLS domain. (a) Color-coded diagram is given of 2° and 3° interactions in the TLS construct in F conformation, (b) color-coded regional map is given of the TLS construct in F conformation, and (c) changes are shown in normalized SHAPE reactivities with increasing [Mⁿ⁺]. To see this figure in color, go online.

Figure 5

SHAPE analysis at the U, I, and F conditions. (a) Normalized SHAPE reactivities are shown at 10 mM [Mg²⁺] + 50 mM [Na⁺] (top), 0.5 mM [Mg²⁺] + 50 mM [Na⁺] (middle), and 25 mM [Na⁺] (bottom). (b–d) Color-coded regional map is given of the (b) F, (c) I, and (d) U conformations. To see this figure in color, go online.

Figure 6

SHAPE reactivity mapped onto literature structure models. (a) SHAPE reactivities obtained at 10 mM [Mg²⁺] + 50 mM [Na⁺] are mapped onto the literature F model. (b) SHAPE reactivities are obtained at 50 mM [Na⁺] mapped onto the literature U model. Colors represent high reactivity (red), moderate reactivity (orange), and low reactivity (black). To see this figure in color, go online.

Figure 7

Comparison of single-molecule folding and SHAPE-guided 2° structure predictions. Fractional populations from single-molecule folding are displayed in a bar graph for all [Mⁿ⁺] conditions explored. Results for the lowest free energy conformer from SHAPE-guided structure predictions are presented above the bar graph. Comparison of both techniques indicates that predictions overestimate the stability of the F conformation at low cation concentrations. To see this figure in color, go online.

Figure 8

SHAPE-directed structure prediction. Circle diagrams (top) and 2° structure representations of all three TLS conformations are shown: (a) predicted F, (b) predicted I, (c) and predicted U conformations. To see this figure in color, go online.

Figure 9

Single-molecule folding guided deconvolution of SHAPE probing. Clean SHAPE profiles after deconvolution with fractional population from single-molecule folding experiments are displayed for F (top), I (middle), and U (bottom). Regions that undergo structural reorganizations are highlighted and color-coded with respect to the 2° structure models on the right. To see this figure in color, go online.