Site-specific protein modification

A method for determining protein-RNA interactions under simulated physiological conditions, the method comprising:

• (a) synthesizing a site specific modified protein, wherein the site specific modified protein comprises a protein modified by replacement of an amino acid, other than lysine or cysteine residues, with an analog of the amino acid, wherein the amino acid analog does not affect a biological activity of the protein;
• (b) subsequently site-specifically labeling the site specific modified protein at the site of the amino acid analog with a donor dye molecule, wherein the site-specific labeling is capable of occurring in the absence of orthogonal protection of nucleophilic side chains of lysine and cysteine;
• (c) providing a RNA molecule labeled with an acceptor dye molecule, wherein the acceptor dye molecule is capable of participating in fluorescence resonance energy transfer with the donor dye molecule;
• (d) measuring the emission of the labeled protein and labeled RNA in (b) and (c) respectively;
• (e) combining the labeled RNA in (c) with the labeled protein in (b) to form a mixture;
• (f) measuring the emission of the mixture in (e);
• and,
• (g) determining the proximity between the donor dye molecule and the acceptor dye molecule.