Preparation of a decadeoxyribonucleotide helix for studies by nuclear magnetic resonance

Abstract

A self-complementary decadeoxyribonucleotide d-CpCpApApGpCpTpTpGpG was chemically synthesized by a procedure based on the phosphotriester approach. This procedure was carefully monitored and appropriately modified to ensure the purity of oligomer components at each step of the synthetic scheme. Extensive use was made of both analytical and preparative high-pressure liquid chromatography to purify and characterize the decamer and its constituent oligonucleotides. The final product (1318 A257 units or 16.5 mumol) was obtained in high purity and sufficient quantity for extensive physical studies by UV, CD, and NMR spectroscopy. Our preliminary results show that at a strand concentration of 1.3 X 10(-5) M and in 0.10 M sodium chloride and 0.01 M sodium phosphate buffer, pH 7.0, the decamer duplex has a Tm at 47 degrees C. The CD spectrum of this decamer duplex is similar to that of B-form DNA. All the resonances of the nonexchangeable base protons of the decamer are well resolved in the 1H NMR spectrum, when the single-stranded form was examined by using a 360-MHz spectrometer and when the duplex form was examined by using a 600-MHz spectrometer. These base proton resonances have been tentatively assigned by using the incremental assignment technique. Although the decamer duplex serves as a substrate for AluI restriction endonuclease, it is not cleaved by HindIII endonuclease.