TRANSPLANTATION OF A 17-AMINO ACID ALPHA-HELICAL DNA-BINDING DOMAIN INTO AN ANTIBODY MOLECULE CONFERS SEQUENCE-DEPENDENT DNA RECOGNITION

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We des cribe the rational design of a DNA-binding antibody, Fab-Ebox, by repl acing a variable segment of the immunoglobulin heavy chain with a 17-a mino acid domain derived from TFEB, a class B basic helix-loop-helix p rotein. DNA-binding activity was studied by electrophoretic mobility-s hift assays in which Fab-Ebox was shown to form a specific complex wit h DNA containing the TFEB recognition motif (CACGTG), Similarities wer e found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Co mparable interference of binding by methylation of cytosine residues i ndicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this stu dy indicate that DNA-binding antibodies of high specificity can be dev eloped by using the modular nature of both immunoglobulins and transcr iption factors.