Abstract
Novelty: Nucleotide analogues having a photoreactive group and a biotinyl group which covalently attach to nucleotide dependent protein binding sites are provided. Protein structurefunction studies are significantly facilitated by receptor binding site directed labelling. The invention has particular application to the characterisation of the active sites of nucleotide dependent enzymes. The use of the method for detection and removal of undesired DNA or RNA from cells (e.g. AIDS viral RNA) is briefly described.
Biology: Analogue modified proteins are detected by avidin-linked peroxidase or alkaline phosphatase methods. The modified protein can be purified by strepavidin-linked or avidinlinked affinity chromatography. After elution of unmodified protein, modified proteins are released and collected from the avidin column by raising the pH to hydrolyze the ester linkage by which the biotin is bound to the ribose hydroxyl group.
Chemistry: The biotin radical is attached to the ribose moiety of the nucleotide through an ester linkage. Upon photo irradiation the biotinylated photoaffinity analogues covalently bond to the nucleotide binding site.