p<i>K</i>_a Shifting in Double-Stranded RNA Is Highly Dependent upon Nearest Neighbors and Bulge Positioning

Shifting of p<i>K</i>_a’s in RNA is important for many biological processes; however, the driving forces responsible for shifting are not well understood. Herein, we determine how structural environments surrounding protonated bases affect p<i>K</i>_a shifting in double-stranded RNA (dsRNA). Using ³¹P NMR, we determined the p<i>K</i>_a of the adenine in an A⁺·C base pair in various sequence and structural environments. We found a significant dependence of p<i>K</i>_a on the base pairing strength of nearest neighbors and the location of a nearby bulge. Increasing nearest neighbor base pairing strength shifted the p<i>K</i>_a of the adenine in an A⁺·C base pair higher by an additional 1.6 p<i>K</i>_a units, from 6.5 to 8.1, which is well above neutrality. The addition of a bulge two base pairs away from a protonated A⁺·C base pair shifted the p<i>K</i>_a by only ∼0.5 units less than a perfectly base paired hairpin; however, positioning the bulge just one base pair away from the A⁺·C base pair prohibited formation of the protonated base pair as well as several flanking base pairs. Comparison of data collected at 25 °C and 100 mM KCl to biological temperature and Mg²⁺ concentration revealed only slight p<i>K</i>_a changes, suggesting that similar sequence contexts in biological systems have the potential to be protonated at biological pH. We present a general model to aid in the determination of the roles protonated bases may play in various dsRNA-mediated processes including ADAR editing, miRNA processing, programmed ribosomal frameshifting, and general acid–base catalysis in ribozymes.