Enzymatic transfer of photo-cross-linkers for RNA-protein photo-cross-linking at the mRNA 5′-cap

Abstract

Photo-cross-linking moieties have proven invaluable for elucidating interactions of biomolecules. While methods for site-specific incorporation of those moieties into proteins have been developed, comparable methods for nucleic acids are lacking. Utilizing the inherent specificity of enzymes, methyltransferases (MTase) exhibiting relaxed cosubstrate specificity in combination with synthetic analogs of S-adenosyl-l-methionine (AdoMet) allow for the precise installation of reporter molecules or affinity tags in various biomolecules. In this chapter, we describe AdoMet analogs with photo-cross-linking moieties that—in combination with an MTase—are ideal for site-specific installation. The workflow for chemo-enzymatic installation of photo-cross-linking moieties at the mRNA cap based on AdoMet analogs is given in detail.