Electron microscopic imaging of supercoiled DNA containing highly biased sequence.

Abstract

DNA conformation of the pUC19 derivatives containing the pyrimidine/purine-biased sequence (pTIR10), highly GC-rich sequence (pTIS303) or short palindrome (pBan1), that had been shown by S1 nuclease assay to conform to unusual DNA structures under superhelical tension, was examined by an electron microscopic imaging. Under the condition of spreading of DNA on the grid that we employed, 20-50% of the molecules were shown to be nicely unfolded and the rest were highly twisted or entangled. About 70% of the unfolded molecules of pTIR10 retained the triplex-like stem of size ranging from 18 nm to 38 nm, as expected from its sequence characteristics. The plasmid pTIS303 showed slightly shorter stem structure than pTIR10, as the pyrimidine/purine-biased stretch in plasmid pTIS303 is shorter than that in pTIR10. The results suggest that the GC-rich segment may be responsible for forming unusual DNA structure in pTIS303.