Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, USA.
PLoS One. 2012;7(11):e48913. doi: 10.1371/journal.pone.0048913. Epub 2012 Nov 8.
Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss- vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of ∼3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells.
刺尾鬣蜥毒素是一种源自南美的响尾蛇 Crotalus durissus terrificus 的毒液的 42 个残基多肽，已被证明是一种细胞穿透蛋白，可靶向染色体，将质粒 DNA 带入细胞，并对活跃增殖的细胞具有特异性。鉴于其作为核酸递药载体的潜在作用，我们在不同的离子条件下详细研究了刺尾鬣蜥毒素与不同长度和碱基组成的单链和双链 DNA 的结合。琼脂糖凝胶电泳和紫外分光光度分析表明，在低离子强度下，刺尾鬣蜥毒素与长链 DNA 的复合物容易聚集和沉淀。这种聚集对于 DNA 的细胞摄取可能很重要，但随着链长的缩短，这种聚集的可能性降低。25 个核苷酸的寡核苷酸没有任何聚集的证据，允许通过荧光猝灭实验确定亲和力和大小。多肽非协同地与 DNA 结合，当它与单链（ss）或双链（ds）DNA 结合时，覆盖约 5 个核苷酸残基。该蛋白对 ss-DNA 与 ds-DNA 的亲和力相当，且与盐浓度成反比。亲和力对[NaCl]依赖性的分析表明，蛋白质与 DNA 之间最多有∼3 个离子相互作用，部分结合亲和力归因于非离子相互作用。检查该蛋白的三维结构表明，残基 31 到 35，Arg-Trp-Arg-Trp-Lys，可能作为潜在的 DNA 结合位点。与全长蛋白相比，含有该序列的六肽显示出较低的 DNA 结合亲和力和盐依赖性，这可能表明更合适的 3D 结构和天然刺尾鬣蜥毒素中存在辅助结合位点。综上所述，这里描述的刺尾鬣蜥毒素-DNA 相互作用的数据可能支持设计更有效的核酸药物传递载体，利用刺尾鬣蜥毒素作为载体，对活跃增殖的细胞具有特异性。
