DNA Pseudoknots with Appropriate Loop Lengths and Sequence Complementary to the Stem form Stabilizing Base-Triplet Stacks

Abstract

Pseudoknots have been found to play important roles in RNA function, such as the critical roles in altering gene expression by inducing ribosomal frameshifting in many viruses and in the 5’ UTR of mRNA as riboswitches. We used a combination of UV spectroscopy and differential scanning calorimetry to investigate the unfolding of DNA pseudoknots that mimic the formation of a local triplet helix found with RNA pseudokkots, explaining −1 ribosomal frameshifting. Specifically, we determined the unfolding thermodynamics for the following DNA set of pseudoknots with sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where the length of the “Tn” loop was varied from n = 5, 7, 9, and 11. The increase in loop length yielded higher TMs, 53C to 59C, and folding enthalpies ranging from −60 kcal/mol to −105 kcal/mol, resulting in a significant stabilization of the pseudoknots, G = −8.5 kcal/mol to −15.9 kcal/mol. We also varied the length of the loop for two sets of control molecules: straight hairpin loops and pseudoknots in which the 5’ loop is not complementary to the stem. Their increased loop length yielded slight changes in both the TMs and folding enthalpies, consistent with a slight decrease in stability with the straight hairpin loops and a slight increase in stability with the pseudoknots. Therefore, the increase in enthalpy, ∼14 kcal/mol per step of two loop thymines, is explained in terms of the formation of a single base-triplet stack. For instance, the pseudoknot with the loop of 9 thymines forms two base-triplet stacks. Supported by Grant MCB-1122029 from NSF and GAANN grant P200A120231 from the U.S. Department of Education.