[en] They have enzymatically synthesized ³H- and ³²P-poly(A,8N₃A) from 8-N₃ADP and radiolabeled ADP, and have used this polynucleotide to photoaffinity label T4 gene 32 protein, as well as several other helix-destabilizing proteins (HDPs). Irradiation of ³²P-³H-poly(A,N₃A) mixtures for short durations produces a covalent complex, seen as a high molecular weight, radioactive band on SDS-polyacrylamide gels. Preliminary experiments on other HDPs, from prokaryotic, eukaryotic and animal viral sources, show analogous results. Several successful control experiments indicate that this system is suitable for binding site localization on ³²P. Single-stranded nucleic acids competitively inhibit photolabeling. The effect of NaCl on photolabeling parallels the salt-dependence of ³²P-poly(A,N₃A) binding. Photolabeling reaches a plateau after ∼1 min, and the formation of the high molecular weight complex parallels the reduction of free ³²P on SDS gels. Staph. nuclease digestion of crosslinked complexes produces a diffuse, radioactive band on SDS gels, migrating just behind free ³²P. When these digested complexes are subjected to reverse-phase HPLC on a C3 Ultrapore column, the nucleic acid ³²P-label is seen to coelute with protein. They are currently employing RP-HPLC methods to locate the label on tryptic peptides of nuclease-digested complexes