Oligodeoxynucleotides interact with recombinant CD4 at multiple sites.

Phosphodiester oligodeoxynucleotides bearing the 5'-alkylating moiety 4-(N-2-chloroethyl-N-methyl)aminobenzylamine specifically modify recombinant soluble CD4 (rsCD4) in solution. This reaction is saturable with respect to the alkylating oligonucleotide reagent. The existence of at least two binding sites, with different affinities, on the rsCD4 molecule, were demonstrated. The values of apparent Kd for the sites are approximately 0.1 and 1 microM. The existence of two sites was confirmed by electrophoretic analysis of the modified protein, in which two distinct gel bands were seen. The modification is inhibited by excess non-alkylating oligonucleotide, as well as by phosphorothioate oligonucleotides. Quantitative estimates of the competition constants (Kc), for the binding of these competitors of the binding of the alkylating oligonucleotide reagent with rsCD4, have been made. By use of this method, several anionic dyes as well as potential anti-HIV therapeutic agents were also demonstrated to interact with rsCD4. Phosphorothioate oligonucleotides also inhibit binding of rsCD4 with the monoclonal antibody L71.1.1 This monoclonal antibody recognizes the CDR3-like loop (D1 domain) of the rsCD4 molecule. Thus, oligonucleotide binding sites exist on two remote regions (i.e. both the CDR2- and CDR3-like loops) of the D1 domain of CD4.