McLane K E, Burton D R, Ghazal P
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1995 May 23;92(11):5214-8. doi: 10.1073/pnas.92.11.5214.
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 describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.
能够与核酸进行序列特异性相互作用的重组抗体代表了一类DNA和RNA结合蛋白，在基础研究和医学领域具有广泛的应用潜力。我们描述了一种DNA结合抗体Fab-Ebox的合理设计，通过用源自TFEB（一种B类碱性螺旋-环-螺旋蛋白）的17个氨基酸结构域替换免疫球蛋白重链的可变片段。通过电泳迁移率变动分析研究了DNA结合活性，结果表明Fab-Ebox与含有TFEB识别基序（CACGTG）的DNA形成了特异性复合物。在TFEB和Fab-Ebox区分含有改变的识别序列的寡脱氧核糖核苷酸的能力方面发现了相似之处。胞嘧啶残基甲基化对结合的可比干扰表明，Fab-Ebox和TFEB都通过沿双链DNA大沟的相互作用与DNA接触。这项研究的结果表明，利用免疫球蛋白和转录因子的模块化性质可以开发出高特异性的DNA结合抗体。
