alexa Assessing the Complex Formation between CrotamineâÂ
ISSN: 2168-9849

Cloning & Transgenesis
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Research Article

Assessing the Complex Formation between Crotamine–a Natural CellPenetrating Peptide–and DNA Using High Sensitive Fluorescence Exclusion Assay

Freitas VJF1*, Alcântara-Neto AS1, Pereira AF1, Campelo IS1, Melo LM1 and Rádis-Baptista G2*
1Laboratory of Physiology and Control of Reproduction, State University of Ceará, Fortaleza-CE, Brazil
2Laboratory of Biochemistry and Biotechnology, Institute of Marine Sciences, Federal University of Ceará, Fortaleza CE, Brazil
Corresponding Authors : Freitas VJF
State University of Ceará
Fortaleza-CE, Brazil
Tel: +55 85 31019861
E-mail: [email protected]
  Baptista GR
Instituto de Ciências do Mar
Universidade Federal do Ceará Av. da Abolição 3207
60165-081, Fortaleza-CE, Brazil
Tel: +55 85 3366-7007
E-mail: [email protected]
Received April 28, 2014; Accepted May 01, 2014; Published September 16, 2014
Citation: Baptista GR, Freitas VJF, Alcântara-Neto AS, Pereira AF, Campelo IS (2014) Assessing the Complex Formation between Crotamine–a Natural Cell-Penetrating Peptide–and DNA Using High Sensitive Fluorescence Exclusion Assay. Clon Transgen 3:128. doi:10.4172/2168-9849.1000128
Copyright: © 2014, Freitas VJF , Baptista GR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Crotamine is a natural cationic polypeptide isolated from the venom of South American rattlesnake (Crotalus durissus terrificus) capable of forming complexes with DNA molecules by means of electrostatic interactions and translocating biological membranes. With such properties, crotamine can act as a transfection agent for several cell types but mainly in a stage of proliferation. However, to improve the biological use of this peptide, it is interesting to better characterize the formation and stability of crotamina-DNA complex. Thus, the present study aimed to analyze the stoichiometry, formation kinetics and protease stability of crotamina-EGFP complex using high sensitive fluorescent methodology, based on fluorophore exclusion assay. The best DNA:crotamine mass proportion for complex formation ranges between 1:10 to 1:40 as mass ratio. In 1:10 group, crotamine was able to complex about 75% of the available DNA as fast as in 20 seconds, and after 10 min of reaction crotamine reached its maximum complexation capacity, being able to complex 80% of total amount of DNA available. Under these conditions, we have determined that 1μg of crotamine can complex 80 μg of circular plasmid DNA (~4700 bp). Crotamine-DNA complex was resistant to proteinase K degradation even after twelve hours of enzymatic incubation. Additionally, the complex was sufficiently stable to trypsin digestion for a long incubation time: more than 73% of crotamine-DNA condensates maintained the integrity after 60 min, whereas 55% was intact after 180 min. Crotamine-DNA complexes were resistant to physiological temperatures, as well as stable for 15 days at 4°C and for two months when stored at -20°C. These results lead us to think that, in the best conditions of preparation proposed here, crotamine-DNA condensates can be used in an efficient and standardized manner as a potential agent of transfection in a transgenesis programme.

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