Transcription And Reverse Transcription Of An Expanded Genetic Alphabet In Vitro And In A Semisynthetic Organism

Through the development of unnatural base pairs that are compatible with native DNA and RNA polymerases and the ribosome, we have expanded the genetic alphabet and enabled in vitro and in vivo production of proteins containing noncanonical amino acids. However, the absence of assays to characterize transcription has prevented the deconvolution of the contributions of transcription and translation to the reduced performance of some unnatural codons. Here we show that RNA containing the unnatural nucleotides is efficiently reverse transcribed into cDNA, and we develop an assay to measure the combined fidelity of transcription and reverse transcription. With this assay, we examine the performance of a wide variety of unnatural codons, both in vitro and in the in vivo environment of a semisynthetic organism. We find that transcription is generally efficient, decoding at the ribosome is generally more challenging, and, correspondingly, sequence-dependent translation efficiency is the origin of variable codon performance.