Proton exchange and base-pair opening kinetics in 5′-d(CGCGAATTCGCG)-3′ and related dodecamers

Abstract

We have used nuclear magnetic resonance (NMR) spectroscopy to measure the lifetimes of individual base-pairs In the palindromic DNA oligonucleotide 5′-d(CGCGAATTCGCG)3′ and in three other dodecamers with symmetrical base substitutions in the sites underlined. The resonances of the hydrogen- bonded imino protons in each of the substituted oligomers In the duplex form have been assigned using one dimensional nuclear Overhauser etfect (1-D NOE) experiments. The lifetimes have been obtained from the dependence of selective longitudinal relaxation times and linewidths of the imino proton resonances on the concentration of base catalyst (Tris) at 25°C and in the presence of 50 mM NaCl. The lifetimes of the central A·T base-pairs have been found to depend on base sequence. They are greatly increased in the dodecamer 5′d(CGCAAATTTGCG)-3′ which contains an A₃ T₃ tract. The lifetimes of the central A base-pairs in 5′-d(CGCGAATTCGCG)-3′, 5′-d(CGCTAATTAGCG)-3′ and 5′-d(CGCCAATTGGCG)-3′ are comparable. In all dodecamers, the lifetime of the A base-pair at the 5′-end of the A_n T_n tract is the shortest. The anomalous opening kinetics of the A·T base-pairs can be correlated to the bending properties of the corresponding sequences.