Biochemical Detection of Cytidine Protonation within RNA

Perturbation of active site functional group pK as is an important strategy employed by protein enzymes to achieve catalysis. There is increasing evidence to indicate that RNAs also utilize functional group pK a perturbation for folding and reactivity. One of the best candidates for a functionally relevant pK a perturbation is the N3 of C (pK a 4.2), which could be sufficiently raised to allow protonation near physiological pH. Here we report the synthesis and use of a series of α-phosphorothioate tagged cytidine analogues whose altered N3 pK as make it possible to efficiently detect functionally relevant protonation events by nucleotide analogue interference mapping. 6-Azacytidine (n6CαS) and 5-fluorocytidine (f 5CαS) both have enhanced acidity at the N3 position (pK a 2.6 and 2.3, respectively) but leave the hydrogen bonding face of C otherwise unaffected. In contrast, pseudoisocytidine (ΨiCαS) is a charge neutral analogue that mimics the hydrogen bonding character of protonated C. To test the utility of these analogues, we characterized the C300+-G97-C277 mutant form of the Tetrahymena group I intron, which is predicted to require C300 protonation for ribozyme folding and reactivity. At neutral to alkaline pHs, C300 was the only site of n6CαS and f 5CαS interference within the intron, yet both interferences were rescued at acidic pH. Furthermore, ΨiCαS substitution at C300 resulted in enhanced activity at alkaline pHs, consistent with the presence of an N3 proton under the pH conditions studied. Interference mapping with these analogues provides an efficient and sensitive means to identify every site within an RNA where cytidine protonation is important for RNA function and may make it possible to identify C's that participate in general acid/base catalysis within ribozyme active sites.