title: DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC), Baltimore, Maryland, USA.
authors.forename: Pei-Chun
authors.initials: PC
authors.lastname: Chen
authors.forename: Mirian A F
authors.initials: MA
authors.lastname: Hayashi
authors.forename: Eduardo Brandt
authors.initials: EB
authors.lastname: Oliveira
authors.forename: Richard L
authors.initials: RL
authors.lastname: Karpel
mesh_terms: D000818:Animals; D003435:Crotalid Venoms / Q000737:chemistry* / Q000378:metabolism; D017839:Crotalus / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D016503:Drug Delivery Systems; D005822:Genetic Vectors; D017434:Protein Structure, Tertiary; D030162:Reptilian Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003435:Crotalid Venoms; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D030162:Reptilian Proteins; D004247:DNA; C010563:crotamine