Synthetic capped mRNAs for cap-specific photo-cross-linking experiments

Abstract

The 7-methylguanosine triphosphate cap present at the 5′ ends of eukaryotic mRNAs plays numerous roles in mRNA expression and metabolism. The identification and studies on cap-binding partners can be significantly advanced using tailored chemical tools such as synthetic cap analogues or RNAs carrying modified cap structures. Here we provide protocols for the production of mRNAs specifically labeled within the 5′ cap with a nucleoside capable of being photo-activated, either 6-thioguanosine or 7-methyl-6-thioguanosine, which can be used in photo-cross-linking experiments to identify or characterize capbinding biomolecules. We also describe a protocol for the cross-linking experiments with capped RNAs to map histone H4 cap-binding pocket.