Abstract
The four natural nucleotides of DNA form base pairs capable of encoding the complex genetic information necessary for all life; additionally, the sequence specific hybridization and enzymatic synthesis of DNA has revolutionized biotechnology. Expansion of the genetic alphabet to include additional, orthogonal nucleotides to work within the context of natural DNA has the potential to greatly expand this essential biopolymer's utility. Here, we detail the three general approaches to the design of unnatural DNA base pairs: alternative hydrogen bonding, shape complementarity, and hydrophobic forces. All of these approaches have been implemented with notable success, but are still limited by DNA polymerase recognition of the unnatural pairs. Thus, we also consider the role of the DNA polymerase and highlight efforts to use alternative DNA polymerases, either natural or engineered.
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