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.

Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support

“…Mesitylenesulfonyl-3-nitrotriazole (MSNT) was purchased from Vega Biochemicals. Anhydrous pyridine was prepared by refluxing previously purified pyridine (5) over calcium hydride chips for several hours followed by distillation onto calcium hydride chips in 5 ml V-vials fitted with Teflon-lined septum caps (Wheaton Scientific). Lyophilized spleen phosphodiesterase was obtained from P.L.…”

“…4) The column is attached to a waste flask and the support is washed with three 2 ml portions of 0.5 M triethylammonium acetate in dimethylformamide; three 2 ml portions of anhydrous pyridine and three 2 ml portions of diethyl ether. 5) The column is fitted with the septum cap and set up in the drying mode under house vacuum for at least 5 min. 6) The support is dried by adding 300 jl of anhydrous pyridine via a gas tight syringe (Hamilton).…”

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2′-O-methylribonucleosides

“…As shown in Table 2 (28)(29)(30)(31)34). The Tms of the duplexes formed with the Sp isomer are also lower than duplexes with the Rp isomer in buffer lacking sodium chloride, although the reduction is somewhat less relative to that observed in 0.1 M sodium chloride buffer.…”

“…In fact in buffer lacking sodium chloride, the Tm of this duplex is 11 C higher than that of 1/DNA. This increased stability is attributed to the reduced charge repulsion between the methylphosphonate backbone of 7 and the diester backbone of the DNA target, versus the greater charge repulsion experienced by 1/DNA which contains two fully charged sugar-phosphate backbones (34).…”

Synthesis and separation of diastereomers of O-(2,2,2-trifluoroethyl)-3′,5′-dinucleoside phosphates

“…Synthetic oligonucleotides that have backbone modifications are useful as model compounds for studying DNA structure and dynamics (1,2), and as probes for elucidating specific interactions of DNA with proteins and enzymes (3)(4)(5). Phosphoric triesters have long been studied as models of these modifications.…”