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 theHoogsteen 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 imidazoletethered 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 imidazoliumtethered 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

Identification of a pKa-regulating motif stabilizing imidazole-modified double-stranded DNA

The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKa(H) are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6 degrees C and is key in modulating the pKa(H) of the imidazolium group. The general features, prerequisites and generic character of the new pKa(H)-regulating motif are described

Versatile synthesis of amino acid functionalized nucleosides via a domino carboxamidation reaction

Functionalized oligonucleotides have recently gained increased attention for incorporation in modified nucleic acid structures both for the design of aptamers with enhanced binding properties as well as the construction of catalytic DNA and RNA. As a shortcut alternative to the incorporation of multiple modified residues, each bearing one extra functional group, we present here a straightforward method for direct linking of functionalized amino acids to the nucleoside base, thus equipping the nucleoside with two extra functionalities at once. As a proof of principle, we have introduced three amino acids with functional groups frequently used as key-intermediates in DNA- and RNAzymes via an efficient and straightforward domino carboxamidation reaction