Advances in RNA structure prediction from sequence: new tools for generating hypotheses about viral RNA structure-function relationships.

Advances in sequencing technology have made abundant RNA sequence information available, but the challenge of how to interpret these data remains. The RNA sequence contains many layers of information. RNA sequences code for proteins and small RNAs, such as microRNAs or transacting small interfering RNAs (siRNAs). RNA encodes information about both structure and function. Viral RNA structures, such as riboswitches, internal ribosome entry sites (75), and panhandles (71), regulate the stages of the viral life cycle, including replication (100), transcription (99), splicing (40, 48), aminoacylation (31, 55), translation (12, 75, 98), and encapsidation (27, 60, 71, 84). Because viral RNAs are structurally dynamic, current prediction methods focusing on a single minimum freeenergy structure may not always identify functionally relevant structures without additional experimental restraints. Because RNA structure determination is often experimentally difficult despite tremendous advances in RNA crystallography, nuclear magnetic resonance spectroscopy, and chemical modification, RNA structure prediction is an important tool for generating hypotheses about structure-function relationships in RNA. RNA structure prediction can be useful for interpreting or designing mutagenesis experiments, identifying conserved structural features, and designing siRNA strategies. This review will briefly outline the basic ideas and assumptions underlying RNA structure prediction, compare different approaches to RNA structure prediction from a user’s perspective, and discuss some applications of RNA structure prediction to viral RNA interference (RNAi) research. The RNA sequence, or primary structure, determines the secondary structure, or pattern of canonical Watson-Crick pairs forming duplexes and irregular regions, such as loops and single-stranded regions. The secondary structure then determines the tertiary structure or overall three-dimensional shape of the molecule. The tertiary structure contains the interaction sites for proteins, other RNA molecules, carbohydrates, or other small molecules and thus determines the quaternary or higher-order structure. The greater thermodynamic stability of RNA helices than that of tertiary interactions makes the RNA folding process hierarchical (35). For example, the favorable free energy involved in forming two stacked base pairs (0.4 to 0.9 kcal/mol per nucleotide) (105) is larger than the favorable free energy to form tertiary interactions, such as ribose zippers or tetraloop-receptor interactions (0.1 to 0.5 kcal/mol per nucleotide) (25, 97). Note that 1.4 kcal/mol is equivalent to 1 order of magnitude in a binding constant at 37°C. The hierarchical nature of RNA folding makes RNA structure prediction a tractable challenge (92).