Elucidation of anti-ssDNA autoantibody BV 04-01 binding interactions with homooligonucleotides.
Binding interactions of various synthetic oligohomonucleotides with anti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding single-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide binding to IgG or SCA resulted in quenching of the protein's tryptophan fluorescence permitting direct assessment of ligand binding under equilibrium conditions. The effect of oligothymidylate length, (dT)n, on tryptophan quenching was evaluated. The equilibrium dissociation constants (Kd) 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 to (dT)3 increased the Kd an order of magnitude. To assess base specificity, the comparative binding of other hexahomonucleotides was examined. Neither (dA)6 nor (dC)6 showed measurable binding, while the dissociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence lifetime quenching data correlated with the steady-state binding results 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 nucleotides was further supported by fluorescence anisotrophy studies with fluorescein-labeled hexadeoxynucleotides. Various levels of tryptophan fluorescence quenching upon titration with oligothymidylates of different 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 binding of oligonucleotides.
Tetin
S Y
SY
Department of Microbiology, University of Illinois, Urbana 61801.
Rumbley
C A
CA
Hazlett
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TL
Voss
E W
EW
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001323:Autoantibodies / Q000737:chemistry / Q000276:immunology*; D004277:DNA, Single-Stranded / Q000276:immunology*; D005454:Fluorescence Polarization; D007700:Kinetics; D008958:Models, Molecular; D009841:Oligonucleotides / Q000276:immunology*; D011071:Poly T / Q000276:immunology; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011071:Poly T; D014364:Tryptophan