Dynamic and Conformational Effects of Structural Perturbations in DNA and RNA Studied by Nuclear Magnetic Resonance and Chemically Induced Dynamic Nuclear Polarization

摘要

The conformation and dynamics of oligonucleotides in solution were studied using several different NMR techniques. The oligonucleotides contain a variety of perturbations; (I) an extra cytosine: dCA sub 3 CA sub 3 G + dCT sub 6 G, (II) an extra adenine: d(CGCAGAATTCGCG) sub 2 , and (III) a G.T mismatch: d(CGTGAATTCGCG) sub 2 . These are compared to the unperturbed duplexes, (IV) dCA sub 6 G + dCT sub 6 G and (V) d(CGCGAATTCGCG) sub 2 . Through the use of nuclear Overhauser effects; the unpaired cytosine in duplex I was found to be extrahelical. Thermodynamic parameters were determined for duplexes I and IV using UV absorption melting curves. The extrahelical cytosine causes a 15 exp 0 C decrease in the duplex melting temperature. The standard free energy for duplex formation at 25 exp 0 C is 2.9 kcal mol exp -1 more positive for the duplex with the extra cytosine. The kinetics for exchange of the imino protons in these oligonucleotide duplexes were studied by saturation recovery techniques. In duplex I, the extra cytosine causes the exchange lifetime for one of the neighboring A.T base pairs to decrease by a factor of two over the other A.T base pairs in the duplex. The extra adenine in duplex II affects the exchange lifetimes throughout the duplex, however, the G.T mismatch in duplex III has a more local effect on the lifetimes. Also presented are solvent accessibility studies of oligonucleotides using Chemically Induced Dynamic Nuclear Polarization (CIDNP). CIDNP was observed for guanine(H8) protons and for guanine(H1') protons in dinucleotides and longer oligonucleoides. (ERA citation 09:008572)