Efforts towards Expansion of the Genetic Alphabet: Pyridone and Methyl Pyridone Nucleobases

Abstract

A non-natural base pair, which is stable in duplex DNA and enzymatically synthesized with high efficiency and selectivity, would greatly expand the utility of nucleic acids, both in terms of their genetic-coding capacity and chemical functionality. Unlike the natural base pairs, a third base pair does not necessarily require H-bonding,[1] and we, [2] and others, [3] have demonstrated that hydrophobic and van der Waals forces can mediate the selective interbase interactions required for basepair stability and synthesis.Our early efforts focused on nucleotides bearing nucleobase analogues with large extended aromatic surfaces. A variety of self-pairs (formed between the same analogue) and heteropairs (formed between different analogues) were identified. These pairs were both stable in duplex DNA and efficiently synthesized by the exonuclease deficient Klenow fragment of DNA polymerase I (Kf).[2a, 4] However, replication was consistently limited by poor extension (i.e. continued primer elongation after synthesis of the non-natural pair), which we reasoned could be the result of interbase intercalation of the large aromatic scaffolds that distort the newly formed primer terminus and prevent its efficient extension. Our second-generation non-natural base pairs were formed between nucleotides that bear small, aromatic nucleobase analogues that were designed to be too small to intercalate and therefore more likely to interact edge on. Although several base pairs have been found that are more efficiently extended by Kf, the rates remain insufficient. [2b, 5] Further-

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.…”

Unnatural Base Pairs for Synthetic Biology

“…However, further replication reactions after PICS : PICS base pairing are terminated because PICS bases overlap with each other, indicating structural change in the DNA duplex. Consequently, they explored more than 100 kinds of unnatural base pairs [19][20][21][22][23][24][25] and succeeded in developing 5SICS : MMO2 and 5SICS : NaM pairs, which are replicable unnatural base pairs in the PCR. [26][27][28] In 2014, they reported the creation of a semi-synthetic organism containing the 5SICS : NaM base pair.…”