Design and systematic study of imidazole based DNAzymes: an integrated NMR and molecular dynamics approach

Proteins are known as the workhorses of the cell and fulfill numerous tasks essential for the cell's survival. One of the most important tasks is their involvement in catalysis. From a thermodynamic point of view, this is made possible by the stabilisation of the transition state, a high-energy reaction state between reactants and products. This stabilization is made possible due to a combination of a wide variety of chemical functionalities inherent to the twenty amino acid building blocks. This variety observed with proteins stands in sharp contrast to the limited structural diversity of DNA and RNA. These biomolecules are optimized for hydrogen bonding and the formation of complementary, predictable helix-like structures. It thus seemed highly unlikely DNA and RNA could ever fulfill the same catalytic functions as proteins. The discovery and subsequent development of natural and synthetic catalytic RNA and DNA (deoxy)ribozymes has overturned this belief. Mainly identified by means of in vitro selection and evolution experiments, RNA/DNA-based catalysts employ a variety of catalytic mechanisms. Nevertheless, despite the numerous successes of the top-down in vitro approach, structural insight in how these RNA- and DNAzymes ultimately achieve catalysis is still lacking and can be considered as one of the main drawbacks for their further development. Inspired by these developments and limitations, the current research project aims to develop DNA-based hydrolases via a more bottom-up approach. Here the stable and predictable nature of the DNA duplex is employed to position one or more imidazole-bearing thymine nucleotide building blocks (TIm) using standard phosphoramidite solid phase chemistry. Via systematic studies using UV-VIS thermal melting experiments, NMR and molecular dynamics simulations, the mutual impact of the modification and the DNA scaffold could be identified. This approach was applied in two major systematic studies focussing on both single and multiple TIm modified DNA systems. In case of the single modified systems a so-called pKaH-regulating motif (figure) has been uncovered where the imidazole modification at position n in the DNA duplex engages in a persistent hydrogen bond interaction with the carbonyl groups of guanine bases at positions n+1 and n+2 residing in the DNA major groove. This interaction in turn contributes in a significant thermal stabilisation of ±6°C and increase of over 1 pKaH unit with respect to other non-interacting modified systems. In addition to its identity and overall features, the possible sequential permutations of this motif were explored as well. Given that a single TIm functionality alone is unlikely to generate any meaningful catalytic activity, the second systematic study focused on the mutual impact of multiple imidazole residues residing in the same system, both in the presence and absence of the interaction motif. Furthermore these systems allowed to confirm that the motif is tolerated when multiple imidazoles are present and relative positioning of the pKaH-regulating motif with respect to the non-interacting imidazole allows to regulate the pKaH value of the second non-interacting imidazole functionality within certain limits as well. The observations and guidelines obtained during these studies should allow to gradually develop more intricate systems that are ultimately able to cleave ester and/or amide bonds in a stereo selective fashion.

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