Since the last common ancestor of all life on earth, the biological diversity has been encoded in a four letter, two base pair genetic alphabet. Expansion of the genetic alphabet to include a fifth and sixth letter than for a third, unnatural base pair not only has immediate utility for a number of applications, such as site-specific oligonucleotide labeling, but also serves as the foundation for an organism with an expanded genetic code. Toward this goal, we have examined a large number of diff