engleski

DNA melting in very low salt: intermediate DNA

We report that DNA molecules in an intermediate state between ssDNA and dsDNA are the majority population in the conditions of low DNA concentration, cDNA=0.05-1mM (basepair) and very low added salt, csalt<0.05 mM. By fluorescence correlation spectroscopy (FCS) we found that the DNA (nucleosomal fragments ~150 basepairs) in that concentration region, at ambient temperature, has the diffusion coefficient Dp value below the values for both ssDNA and native dsDNA of similar polymerization degree N. Thus, the DNA population specific to the very low added salt conditions can not be a simple mix of ssDNA strands which result from the melting and of original dsDNA fragments. We suggest that the DNA is, due to the unscreened Coulomb potential inherent to the low salt conditions, stabilized in a partly denatured, intermediate state. This result is rationalized through the well known concept of fluctuational openings (DNA bubbles) which occur below and along dsDNA melting transition. In our case, due to very low salt, DNA is locally untwisted and stretched due to the electrostatic repulsion of unscreened phosphate charges - separated DNA strands in the bubble are rigid and linear. Thus, the intermediate DNA also forms a rigid rod, like the original dsDNA, however, due to ssDNA segments, stretched for about 10 %, judged from the difference in Dp. This value also indicates that about half of DNA sequence is open, since ssDNA contour is 20% longer than the contour of dsDNA of similar N.

DNA melting; intermediate DNA; conductometry; fluorescence correlation spectroscopy

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