质粒DNA超螺旋构象的激光喇曼光谱研究

将pBR322重组质粒DNA纯化,经琼脂糖凝胶电泳鉴定其主要具共价闭合环状空间构象。对此制备物进行激光喇曼散射光谱分析,发现在表征其二级结构为B型的特征模之外,还有另外二个表征磷酸脱氧核糖主链骨架振动状态的特征模854和1083cm~(-1)。本文对此进行探讨,认为这二个特征模与闭合环状DNA分子的超螺旋状态有关,可作为质粒DNA三级结构的特征模。碱基堆积状态分析表明,超螺旋的存在使分子中脱氧胸苷的堆积反应活性增强,并使AT碱基间Hoogsteen型氢键有相当数量的破坏,导致反映脱氧胸苷参与氢键组成的二个基团振动状态的特征模1378cm~(-1)产生相对于线性DNA分子的明显减色及脱氧胸苷羰基双键振动模向高波数偏移。

LASER RAMAN SPECTRAL ANALYSIS OF SUPERHELICAL SPATIAL CONFORMATION ON PLASMID DNA

The pBR322 recombinant plasmids DNA products, which purified by phenol-extract and equilibrium centrifugation in CsCl -ethidium bromide gradients, have been shown as mainly spatial conformation of covalently closed circular DNA (i. e. cccDNA) by agarose gel electrophoresis for homogeneity detection. Laser Raman spectra in the region 450 - 1750cm-1 have been obtained for the circular double-stranded plasmids DNA molecule in an aqueous solution, indicating not only contain those marker peaks of secondary structure as in conventional B-form of DNA, but also present both 854 and 1083 cm-1 diagnostic bands of reflecting the vibration state of deoxyribosyl phosphodiester backbone. The present analysis have demonstrated relationships between the superhelical state for cccDNA and the two conformational marker bands that can be considered as the tertiary structure marker on plasmids DNA. Because of markedly Raman hypochromicity of charecter band 1378cm-1 of dT in contrast to liear DNA molecule, as well as the carbonyl double bond vibration line of dT have shifted to higher wave number position, the base stacking analysis represent occurrence of both increase in staking reaction activity of dT, and injury of a number of the Hoogsteen hydrogen bond between dA and dT with exist supercoil structure in cccDNA.