Despite the biological importance of non-coding RNA, their structural characterization remains challenging. Making use of the rapidly growing sequence databases, we analyze nucleotide coevolution across homologous sequences via Direct-Coupling Analysis to detect nucleotide-nucleotide contacts. For a representative set of riboswitches, we show that the results of Direct-Coupling Analysis in combination with a generalized Nussinov algorithm systematically improve the results of RNA secondary structure prediction beyond traditional covariance approaches based on mutual information. Even more importantly, we show that the results of Direct-Coupling Analysis are enriched in tertiary structure contacts. By integrating these predictions into molecular modeling tools, systematically improved tertiary structure predictions can be obtained, as compared to using secondary structure information alone.

frameshifting. This is a common mechanism found in viruses, where there is a change in the reading frame allowing for different mRNAs to be translated. This arises when a pseudoknot initiates the frameshift at the slippery sequence. The high-efficiency of frameshifting may be attributed to the formation of a local triplex within the pseudoknot, resulting in an increased interaction with the ribosome. Furthermore, the overall structure and conformation of a pseudoknot results from the actual length of the loops, number of stem base pairs and stem-loop interactions. We have previously reported that the higher enthalpy contribution of the DNA pseudoknot with sequence, d(TCTCTT 11 AAAAAAAAGAGAT 5 TTTTTTT) (Psk-11), is due to the formation of three base-triplet stacks. This takes place because the ''T 11 '' loop is complementary to the stem sequence. In this work, we use a combination of UV spectroscopy and differential scanning calorimetry (DSC) to investigate the unfolding thermodynamics of two pseudoknot analogs, one with a ''dU 11 '' loop (Psk-11U) and the second one with a RNA sequence r(UCUCUU 11 AAAAAAAA-GAGAU 5 UUUUUUU) (RNAPsk-11). Both analogs form intramolecular pseudoknots. Their stability and unfolding enthalpies both follow the order: RNAPsk-11 > Psk-11 > Psk-11U. The resulting thermodynamic profiles will be discussed in terms of specific base-pair and base-triplet stacking contributions. Supported by Grant MCB-1122029 from NSF.