Physicochemical studies on polydeoxyribonucleotides containing defined repeating nucleotide sequences

Abstract

Absorbance-temperature transition, analytical buoyant density, and circular dichroism studies are reported on a variety of high molecular weight doublestranded DNA polymers containing defined repeating nucleotide sequences. These studies provide further evidence that the structure of a DNA is dictated by its primary nucleotide sequence. The DNA's studied include three homopolymer pairs, five repeating dinucleotide DNA's and four repeating trinucleotide DNA's. Equilibrium absorbance-temperature studies over a 100-fold salt concentration range reveal the following: (1) DNA's which have the same base composition but different nucleotide sequences (sequence isomers) do not show identical helixcoil transitions. Sequence isomeric DNA's † † For a description of pairs of sequence isomeric DNA's see Figure 12. have T m values differing by as much as 9 deg. C. (2) For sequence isomeric polymers, the DNA containing both purines and pyrimidines on each of the complementary strands is more stable than the isomer containing only purines on one strand and only pyrimidines on the complementary strand. This generalization is valid for 80% of the cases examined. (3) All polymers undergo a single helix-random coil transition over a narrow temperature range at all salt concentrations studied. (4) The transitions are virtually completely reversible, particularly at salt concentrations above 0.1 m-Na +. (5) Poly d(I-C)·poly d(I-C) ‡ shows a marked increase in the breadth of the melting transitions as the salt concentration is raised. A similar behavior for poly d(A-T)·poly d(A-T) has been reported. Density gradient analyses were performed on the DNA polymers. The ordering of polymer densities in Cs 2SO 4 solution is similar to that previously reported for CsCl gradients. In comparing sequence isomeric DNA's (for example poly dA · poly dT and poly d(A-T)·poly d(A-T)), the DNA which is more thermostable is always less dense in a CsCl gradient analysis. Circular dichroism studies on eleven different DNA polymers show that nucleotide sequence strongly affects spectra. Semi-empirical nearest-neighbor calculations are used to attempt to predict some spectra. Only moderate agreement between calculated and observed spectra is found, thus providing further evidence that the configuration of all polymers is not the same. However, the inclusion of certain approximations facilitates the prediction of the circular dichroism spectrum of naturally occurring DNA from the experimentally obtained polymer spectra. The calculated spectrum for salmon sperm DNA is in excellent agreement with its observed spectrum. The synthesis and characterization of a new DNA polymer, poly d(G-C)·poly d(G-C), is described. The possible biological implications of the influence of nucleotide sequence on DNA physicochemical properties are discussed.