ELUCIDATION OF ANTI-SSDNA AUTOANTIBODY BV-04-01 BINDING INTERACTIONS WITH HOMOOLIGONUCLEOTIDES

Binding interactions of various synthetic oligohomonucleotides with an ti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding si ngle-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide bind ing to IgG or SCA resulted in quenching of the protein's tryptophan fl uorescence permitting direct assessment of ligand binding under equili brium conditions. The effect of oligothymidylate length, (dT)n, on try ptophan quenching was evaluated. The equilibrium dissociation constant s (K(d)) for the binding of (dT)6 and (dT)8 were the same [(1.3 +/- 0. 02) x 10(-7) M], while decreasing the length of the oligothymidylate t o (dT)3 increased the K(d) an order of magnitude. To assess base speci ficity, the comparative binding of other hexahomonucleotides was exami ned. Neither (dA)6 nor (dC)6 showed measurable binding, while the diss ociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence lifetime quenching data correlated with the steady-state binding resu lts and indicated that the quenching process contains both dynamic and static components. The ability of BV 04-01 to bind (dT)6 and (dG)6 nu cleotides was further supported by fluorescence anisotropy studies wit h fluorescein-labeled hexadeoxynucleotides. Various levels of tryptoph an fluorescence quenching upon titration with oligothymidylates of dif ferent length, as well as the similar affinities for (dT)6 and (dG)6, supported the concept that the groove-type binding pocket in BV 04-01 consists of binding subsites that cooperatively adapt for efficient bi nding of oligonucleotides.