Abstract
The complex formed between32P-labeled (dT)15and a hexahistidine (6-His)-tagged anti-single-stranded DNA (ssDNA) Fab, DNA-1, was trapped by addition of nickel-chelating nitrilotriacetic acid (Ni–NTA) agarose that led to efficient separation of bound ligand from free. High stability of the immobilized complex (half-life of 4 h) and low nonspecific binding of [32P](dT)15allowed for a rapid estimation of the dissociation constant (Kd) and was found to be ∼130 nm. Oligonucleotide bound DNA-1 preimmobilized on Ni–NTA agarose with the sameKdas the Fab/(dT)15complex formed in solution, indicating that the interaction of the 6-His tag with the resin did not interfere with binding. Addition of unlabeled (dT)15led to a fast exchange with bound [32P](dT)15. Mutant versions of DNA-1 were also examined and results obtained were in agreement with data from equilibrium gel filtration and fluorescence titration [A. A. Komissarov, M.J. Calcutt, M.T. Marchbank, E.N. Peletskaya, and S.L. Deutscher (1996)J. Biol. Chem.271, 12241–12246]. These results demonstrate that the Ni–NTA assay is an efficient and accurate method to examine 6-His-tagged protein–nucleic acid complexes. Furthermore, a competition modification of this assay may be used for detection of anti-ssDNA antibodies in serum.
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