Unnatural Base Pairs to Expand the Genetic Alphabet and Code

Abstract

The genetic instructions for all life, from humans to microorganisms, going back to the last common ancestor have been encoded in DNA through the selective pairing of guanine (G) with cytosine (C) and adenine (A) with thymine (T). The addition of nucleotide letters to the genetic alphabet to form a third base pair would increase the information storage potential of this central biological system, potentially enabling many applications in basic as well as applied research. Expansion of the genetic alphabet would also contribute to our understanding of life itself by addressing the uniqueness of the natural purines and pyrimidines and the underlying forces that dictate their structure and function. Different approaches to developing additional nucleotide letters for the genetic alphabet have been undertaken. One approach relies on designing nucleotides that pair using hydrophobic and packing forces to provide selective and orthogonal base pairing, and this has yielded a large number of candidates, which through successive rounds of evaluation and optimization has resulted in several unnatural base pairs (UBPs) that are fully compatible with natural nucleic acid biochemistry. These UBPs are replicated and transcribed by native enzymes with remarkable efficiency and fidelity and can be incorporated into unnatural codons to direct protein production at the ribosome, including in living organisms. This chapter presents an overview of the discovery and characterization of these UBPs, their deployment in an expanded genetic alphabet and code in living cells, and their use to develop novel protein therapeutics.