D-CpCpGpG and d-GpGpCpC self-complementary duplexes: Nmr studies of the helix-coil transition.

Abstract

AbstractWe have monitored the helix‐coil transition of the self‐complementary d‐CpCpGpG and d‐GpGpCpC sequences (20mM strand concentration) at the base pairs, sugar rings, and backbone phosphates by 360‐MHz proton and 145.7‐MHz phosphorus nmr spectroscopy in 0.1M phosphate solution between 5 and 95°C. The guanine 1‐imino Watson‐Crick hydrogen‐bonded protons, characteristic of the duplex state, are observed below 10°C, with solvent exchange occurring by transient opening of the tetranucleotide duplexes. The cytosine 4‐amino Watson‐Crick hydrogen‐bonded protons resonate 1.5 ppm downfield from the exposed protons at the same position in the tetranucleotide duplexes, with slow exchange indicative of restricted rotation about the C‐N bond below 15°C. The guanine 2‐amino exchangeable protons in the tetranucleotide sequence exhibit very broad resonances at low temperatures and narrow average resonances above 20°C, corresponding to intermediate and fast rotation about the C‐N bond, respectively. Solvent exchange is slower at the amino protons compared to the imino protons since the latter broaden out above 10°C. The well‐resolved nonexchangeable base proton chemical shifts exhibit helix‐coil transition midpoints between 37 and 42°C. The transition midpoints and the temperature dependence of the chemical shifts at low temperatures were utilized to differentiate between resonances located at the terminal and internal base pairs while the H‐5 and H‐6 doublets of individual cytosines were related by spin decoupling studies. For each tetranucleotide duplex, the cytosine H‐5 resonances exhibit the largest chemical shift change associated with the helix‐coil transition, a result predicted from calculations based on nearest‐neighbor atomic diamagnetic anisotropy and ring current contributions for a B‐DNA duplex. There is reasonable agreement between experimental and calculated chemical shift changes for the helix‐coil transition at the internal base pairs but the experimental shifts exceed the calculated values at the terminal base pairs due to end‐to‐end aggregation at low temperatures. Since the guanine H‐8 resonances of the CpCpGpG and d‐CpCpGpG sequences exhibit upfield shifts of 0.6–0.8 and <0.1 ppm, respectively, on duplex formation, these RNA and DNA tetranucleotides with the same sequence must adopt different base‐pair overlap geometries. The large chemical shift changes associated with duplex formation at the sugar H‐1′ triplets are not detected at the other sugar protons and emphasize the contribution of the attached base at the 1′ position. The coupling sum between the H‐1′ and the H‐2′ and H‐2″ protons equals 15–17 Hz at all four sugar rings for the d‐CpCpGpG and d‐GpGpCpC duplexes (25°C), consistent with a C‐3′ exo sugar ring pucker for the deoxytetranucleotides in solution. The temperature dependent phosphate chemical shifts monitor changes in the ω,ω′ angles about the O‐P backbone bonds, in contrast to the base‐pair proton chemical shifts, which monitor stacking interactions.