Determinants of Unnatural Nucleobase Stability and Polymerase Recognition

Abstract

Six new unnatural nucleobases have been synthesized and characterized in terms of stability and selectivity of self-pairing in duplex DNA and efficiency and fidelity of self-pairing during polymerase-mediated replication. Each nucleobase has a conserved ring structure but differs from the others in its specific pattern of substitution with oxygen and sulfur atoms. Heteroatom derivatization within the conserved scaffold is shown to have only moderate effects on unnatural self-pair synthesis by the polymerase; larger effects were observed on the thermal stability and polymerase-mediated extension of the self-pairs. The largest effects of heteroatom substitution were on the stability and synthesis of mispairs between the unnatural and natural bases. Certain heteroatom substitutions were found to have a general effect while others were found to have effects that were specific for a particular unnatural or natural base. The data are useful for designing stable and replicable third base pairs and for understanding the contributions of nucleobase shape, polarity, and polarizability to the stability and replication of DNA.

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

“…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).…”

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.…”

Self‐Replication in Chemistry and Biology

“…13-5) as well as some fluoroaromatic analogues [68] and evaluated them as potential self-pairing nucleosides [69,70]. Various of the analogues prepared are also recognized with reasonable selectivity by DNA polymerases [49,68,71].…”