DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier

• Published in: PloS one 2012-11, Vol.7 (11), p.e48913
• Main Authors: Chen, Pei-Chun, Hayashi, Mirian A F, Oliveira, Eduardo Brandt, Karpel, Richard L
• Format: Article
• Language: English
• Subjects: Acids; Affinity; Agglomeration; Amino acids; Analysis; Animals; Antimicrobial agents; Binding sites; Biochemistry; Biology; Chains; Chemistry; Chromosomes; Crotalid Venoms - chemistry; Crotalid Venoms - metabolism; Crotalus - metabolism; Crotalus durissus terrificus; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - metabolism; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Drug carriers; Drug delivery; Drug Delivery Systems; Drug development; Fluorescence; Gel electrophoresis; Genes; Genetic research; Genetic Vectors; Heparan sulfate; Inspection; Ionic interactions; Ionic strength; Ligands; Mammals; Nucleotide sequence; Oligonucleotides; Plasmids; Polypeptides; Protein structure; Protein Structure, Tertiary; Proteins; Quenching; Rattlesnakes; Reptilian Proteins - chemistry; Reptilian Proteins - metabolism; Residues; Salts; Sodium chloride; Spectrophotometry; Surface active agents; Venom
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0048913

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.