Keller Michael, Tagawa Toshiaki, Preuss Monika, Miller Andrew D
Department of Chemistry, Imperial College Genetic Therapies Centre, Flowers Building, Armstrong Road, South Kensington, London SW7 2AZ, UK.
Biochemistry. 2002 Jan 15;41(2):652-9. doi: 10.1021/bi0156299.
Cationic peptides containing Lys and Arg residues interact with DNA via charge-charge interactions and are known to play an important role in DNA charge neutralization and condensation processes. In this paper, we describe investigations of the interaction of the cationic adenovirus core complex peptide mu with a dodecameric ODN (12 bp) and pDNA (7528 bp) using a combination of fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, and photon correlation spectroscopy. Comparisons are made with protamine, a cationic peptide well-known for DNA charge neutralization and condensation. Equilibrium dissociation constants are derived independently by both CD and ITC methods for the interaction between protamine or mu with pDNA (K(d) = 0.6-1 microM). Thermodynamic data are also obtained by ITC, indicating strong charge-charge interactions. The interaction of protamine with pDNA takes place with decreasing entropy (-28.7 cal mol(-1) K(-1)); unusually, the interaction of mu with pDNA takes place with increasing entropy (Delta S degrees (bind) = 11.3 cal mol(-1) K(-1)). Although protamine and mu appear to destabilize pDNA double helix character to similar extents, according to CD thermal titration analyses, PCS studies show that interactions between mu and pDNA result in the formation of significantly more size-stable condensed particles than protamine. The enhanced flexibility and size stability of mu-DNA (MD) particles (80-110 nm) compared to protamine counterparts suggest that MD particles are ideal for use as a part of new nonviral gene delivery vectors.
含有赖氨酸和精氨酸残基的阳离子肽通过电荷-电荷相互作用与DNA相互作用,并且已知在DNA电荷中和及凝聚过程中发挥重要作用。在本文中,我们描述了使用荧光光谱、圆二色光谱、等温滴定量热法和光子相关光谱法相结合的方法,对阳离子腺病毒核心复合肽μ与十二聚体寡核苷酸(12 bp)和质粒DNA(7528 bp)之间相互作用的研究。将其与鱼精蛋白进行了比较,鱼精蛋白是一种以DNA电荷中和及凝聚而闻名的阳离子肽。通过CD和ITC方法独立推导了鱼精蛋白或μ与质粒DNA相互作用的平衡解离常数(K(d)=0.6 - 1 microM)。ITC还获得了热力学数据,表明存在强烈的电荷-电荷相互作用。鱼精蛋白与质粒DNA的相互作用伴随着熵的降低(-28.7 cal mol(-1) K(-1));不同寻常的是,μ与质粒DNA的相互作用伴随着熵的增加(ΔS°(结合)=11.3 cal mol(-1) K(-1))。尽管根据CD热滴定分析,鱼精蛋白和μ似乎使质粒DNA双螺旋特征不稳定的程度相似,但PCS研究表明,μ与质粒DNA之间的相互作用导致形成的尺寸稳定的凝聚颗粒比鱼精蛋白显著更多。与鱼精蛋白对应的颗粒相比,μ-DNA(MD)颗粒(80 - 110 nm)增强的柔韧性和尺寸稳定性表明,MD颗粒非常适合用作新型非病毒基因递送载体的一部分。