Design, synthesis and association study of universal readers for recognition tunneling

For reading DNA bases more accurately, a series of nitrogen-containing aromatic heterocycles have been designed and synthesized as candidates of universal reader to interact with all naturally occurring DNA nucleobases by hydrogen bonding interaction and eventually is used to read DNA by recognition tunneling. These recognition molecules include 6-mercapto-1H-benzo[d]imidazole-2-carboxamide, 5-(2-mercaptoethyl)-1H-imidazole-2-carboxamide, 5-(2-mercaptoethyl)-4H-1,2,4-traizole-3-carboxamide and 1-(2-mercaptoethyl)-1H-pyrrole-3-carboxamide. Their formation of hydrogen bonding complexes with nucleobases was studied and association constants were measured by proton NMR titration experiments in deuterated chloroform at room temperature. To do so, the mercaptoethyl chain or thiol group of these reading molecules was replaced or protected with the more lipophilic group to increase the solubility of these candidates in CDCl3. The 3' and 5' hydroxyl groups of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC) and thymidine (dT) were protected with tert-butyldimethylsilyl (TBDMS) to eliminate hydrogen bonding competition from the hydroxyl protons with these candidates as well as to increase the solubility of the nucleosides in CDCl3 for NMR titration experiment. Benzimidazole and imidazole containing readers exhibited the strongest H-bonding affinity towards DNA bases where pyrrole containing reader showed the weakest affinity. In all cases, dG revealed the strongest affinity towards the readers while dA showed the least.

The molecular complex formation in aqueous solution was studied by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry. The formation of both 1:1 and 2:1 complexes between one or two reading molecules and a DNA nucleotide were observed by ESI mass. A series of amino acids and carbohydrates were also examined by mass spectrometry to show the formation of non-covalent complexes with imidazole reader in aqueous solution. The experimental results were compared by calculating energies of ground state conformers of individual molecules and their complexes using computer modeling study by DFT calculations. These studies give insights into the molecular interactions that happen in a nanogap during recognition tunneling experiments.