Enzymatic Incorporation of a Coumarin-Guanine Base Pair

Previous expansions beyond nature's preferred base-pairing interactions have utilized either nonpolar shape-fitting interactions or classical hydrogen bonding. Reported here is a hybrid of these systems. By replacing a single N-H with C-H at a Watson-Crick interface, the design space for new drug candidates and fluorescent nucleobase analogues is dramatically expanded, as demonstrated here by the new, highly fluorescent deoxycytidine mimic 3-glycosyl-5-fluoro-7-methoxy-coumarin-2'-deoxyribose (dC^C ). dGTP is selectively incorporated across from a template dC^C during enzymatic DNA synthesis. Likewise, dC^C is selectively incorporated across from a template guanine when dC^C is provided as the triphosphate dC^C TP. DNA polymerase I (Klenow fragment) exhibited about a 10-fold higher affinity for dC^C TP than dCTP, allowing selective incorporation of dC^C in direct competition experiments. These results demonstrate that a single C-H can replace N-H at a Watson-Crick-type interface with preservation of functional selectivity and enhanced activity.