Analysis of the Structure and Stability of a Backbone-Modified Oligonucleotide: Implications for Avoiding Product Inhibition in Catalytic Template-Directed Synthesis

Abstract

The structural and thermodynamic origins of the destabilization of a backbone-modified DNA duplex 1, formed between d(CpGpTNTpGpC), containing a single aminoethyl group (−CH2−CH2−NH2+−) in place of the phosphodiester (−O−PO2-−O−) linkage of the central TT dimer, and d(GpCpApApCpG) are investigated. Analyses for the corresponding native duplex and two other related structural analogues of duplex 1 have been compared. Duplex 1 shows a cooperative thermal melting transition that is consistent with a two-state process. At a 2 mM concentration, the melting temperature of duplex 1 is reduced by 17 °C from the native duplex, and this decrease in stability is further assigned to an unfavorable decrease in enthalpy of 7 kcal mol-1 and a favorable increase in entropy of 15 eu mol-1. NMR structural analysis shows that the modified duplex 1 still adopts a canonical B-DNA conformation with Watson−Crick base pairing preserved; however, the CH2 group that replaces the native PO2- group in the modified backbone is flexib...