Stability and Polymerase Recognition of Pyridine Nucleobase Analogues: Role of Minor‐Groove H‐Bond Acceptors

Abstract

In the groove: In an effort to develop unnatural base pairs, pyridyl nucleoside analogues were synthesized and characterized (see structures). An α‐glycosidic nitrogen atom provides an H‐bond acceptor that does not significantly facilitate pairing with natural nucleobases. However, it forms minor‐groove H‐bonds with water molecules and DNA polymerases that optimize the stability and replication, respectively, of the unnatural base pair.

Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

“…Consistent with previous studies [20][21][22][23][24] , primers bearing a hydrogen bond acceptor in the minor groove (i.e. d2OMe, dMMO2, and d4MP) were extended more efficiently than those bearing either hydrogen or a methyl group in the minor groove (dMM3 or dDM5), regardless of the templating nucleobase.…”

“…42 We have repeatedly observed the same phenomenon in unnatural scaffolds. [18][19][20][21][22] Here, this idea is further supported by the significantly decreased efficiency of extension of primers terminating in either dMM3 or dDM5. Despite the importance of this H-bond, the strength of the interaction appears to be less important; within the dSICS scaffold, the oxygen and sulfur substituted analogs are extended essentially equivalently; within the dMMO2 scaffold, the methoxy and carbonyl variants are extended similarly as well.…”

“…8,9,11,14,15,20,22,[28][29][30][31][32][33][34] From these, 60 were collected ( Figure 2) and their phosphoramidites incorporated into the 3' end of a 24-mer primer oligonucleotide and at the 24 th position of a complementary 45-mer template oligonucleotide. Hybridization of any given primer strand with any template strand results in an unnatural primer terminus (dX:dY, where dX is the primer nucleobase and dY is the template nucleobase).…”

“…extension, tends to be relatively inefficient and generally limits the utility of these base pairs. Recent efforts to modify either the nucleobases [20][21][22][23][24] or the DNA polymerase 25 have significantly improved the rate of extension of unnatural base pairs and have demonstrated that efficient extension by the exonuclease deficient Klenow fragment of E. coli DNA Pol I (Kf) likely requires a minimally distorted primer terminus with a suitably positioned minor groove H-bond acceptor in the primer nucleobase. Unfortunately, these strategies have yet to yield a viable base pair candidate as the modifications that facilitate extension have also been found to limit synthesis and increase mispairing.…”

“…Unfortunately, these strategies have yet to yield a viable base pair candidate as the modifications that facilitate extension have also been found to limit synthesis and increase mispairing. 20- 22 Thus, rational approaches to base pair discovery have been limited by the conflicting demands of efficient synthesis and extension. In contrast to approaches based on rational design, screening-based approaches are not limited by our incomplete understanding of DNA stability and replication, and they can potentially identify both the determinants of efficient replication as well as unanticipated unnatural base pairs.…”