P K _a shifting in double-stranded RNA is highly dependent upon nearest neighbors and bulge positioning

Shifting of pK_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 pK_a shifting in double-stranded RNA (dsRNA). Using ³¹P NMR, we determined the pK_a of the adenine in an A⁺·C base pair in various sequence and structural environments. We found a significant dependence of pK_a on the base pairing strength of nearest neighbors and the location of a nearby bulge. Increasing nearest neighbor base pairing strength shifted the pK_a of the adenine in an A⁺·C base pair higher by an additional 1.6 pK_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 pK_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 pK_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.