Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop.

U6 RNA contains a 1 × 2-nt internal loop that folds and unfold during spliceosomal assembly and activation. The 1 × 2 loop consists of a C67•A79 base pair that forms an additional hydrogen bond upon protonation, C67•A+79, and uracil (U80) that coordinates the catalytically essential magnesium ions. We designed a series of RNA and DNA constructs with a 1 × 2 loop sequence contained in the ISL, and its modifications, to measure the thermodynamic effects of protonation and magnesium binding using UV-visible thermal denaturation experiments. We show that the wild-type RNA construct gains 0.43 kcal/mol in 1 M KCl upon lowering the pH from 7.5 to 5.5; the presence of magnesium ions increases its stability by 2.17 kcal/mol at pH 7.5 over 1 M KCl. Modifications of the helix closing base pairs from C-G to U•G causes a loss in protonation-dependent stability and a decrease in stability in the presence of magnesium ions, especially in the C68U construct. A79G single-nucleotide bulge loop construct showed the largest gain in stability in the presence of magnesium ions. The DNA wild-type construct shows a smaller effect on stability upon lowering the pH and in the presence of magnesium ions, highlighting differences in RNA and DNA structures. A U6 RNA 1 × 2 loop sequence is rare in the databases examined.