Affinity electrophoresis for monitoring terminal phosphorylation and the presence of queuosine in RNA. Application of polyacrylamide containing a covalently bound boronic acid.
An affinity electrophoretic method has been developed to study the state of terminal phosphorylation of RNAs and the presence of the hypermodified base Q in tRNA. It is based on the copolymerization of acryloylaminophenylboronic acid into standard polyacrylamide gels and the interaction of this derivative with free cis-diol groups present in the RNA. In the case of terminal phosphorylation, free ribose groups are present either as such, or may be introduced by enzymatic reactions specific for a particular phosphorylation pattern (e.g. using T4 RNA ligase or guanylyltransferase). Additionally, tRNA species containing the Q base may be resolved from Q-lacking tRNAs by boronate affinity electrophoresis. The introduction of a non-destructive, one-step electrophoretic procedure not only offers an alternative to classical analytical methods, but also provides a means of isolating such populations of RNAs for which other methods are unavailable or are less convenient.
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D020106:Acrylamide; D000178:Acrylamides; D001897:Boronic Acids; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006151:Guanosine / Q000031:analogs & derivatives*; D009704:Nucleoside Q / Q000032:analysis*; D010710:Phosphates / Q000032:analysis*; D010766:Phosphorylation; D012313:RNA / Q000032:analysis*; D012315:RNA Caps / Q000032:analysis; D012323:RNA Processing, Post-Transcriptional
D000178:Acrylamides; D001897:Boronic Acids; D010710:Phosphates; D012315:RNA Caps; D006151:Guanosine; D020106:Acrylamide; D009704:Nucleoside Q; D012313:RNA