Biochemical detection of adenosine and cytidine ionization within RNA by interference analysis.
Perturbation of active site functional group pKas is an important strategy employed by protein enzymes to achieve catalysis. There is increasing evidence to indicate that RNAs also utilize functional group pKa perturbation for folding and reactivity. The two best candidates for a functionally relevant pKa perturbation are the N3 of C (pKa 4.2) and the N1 of A (pKa 3.5), either of which could be sufficiently raised to allow protonation near physiological pH. Here we report the synthesis and use of a series of alpha-phosphorothioate tagged cytidine and adenosine analogs whose altered pKas make it possible to efficiently detect functionally relevant protonation events by Nucleotide Analog Interference Mapping. This approach has been used to detect ionization events in several catalytic RNAs, including the group I intron, the Hepatitis Delta Virus (HDV), the hairpin and the Varkud Satellite (VS) ribozymes. The active site residue of the VS ribozyme appears to be ionized in the course of the reaction pathway, which may be indicative of a general acid or base mechanism for catalysis by this RNA.
D000241:Adenosine / Q000737:chemistry*; D003562:Cytidine / Q000737:chemistry*; D007477:Ions; D034622:RNA Interference
D007477:Ions; D003562:Cytidine; D000241:Adenosine