Tethered imidazole mediated duplex stabilization and its potential for aptamer stabilization

Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pK(a). Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and the Hoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pK(a)-motif responsible for these duplex-stabilizing and pK(a)-modulating properties. Here, the robustness and utility of this pK(a)-motif was explored by introducing multiple imidazole-tethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pK(a)-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pK(a) values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazolium-tethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the l-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers.