Improved synthesis of the unnatural base NaM, and evaluation of its orthogonality in in vitro transcription and translation

Unnatural Base Pairs (UBP) promise to diversify cellular function through expansion of the genetic code. Some of the most successful UBPs are the hydrophobic base pairs 5SICS:NaM and TPT3:NaM developed by Romesberg. Much of the research on these UBPs has emphasized strategies to enable their efficient replication, transcription and translation in living organisms. These experiments have achieved spectacular success in certain cases; however, the complexity of working in vivo places strong constraints on the types of experiments that can be done to optimize and improve the system. Testing UBPs using in vitro, on the other hand, offers advantages including minimization of scale, the ability to precisely control the concentration of reagents, and simpler purification of products. Here we investigate the orthogonality of NaM-containing base pairs in transcription and translation, looking at background readthrough of NaM codons by the native machinery. We also describe an improved synthesis of NaM UBP triphosphate and a new assays for testing the purity of NaM containing RNAs.