[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 450-1750 cm-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 relationship 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.