Transcription, Reverse Transcription, and Analysis of RNA Containing Artificial Genetic Components

Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide letters. We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H) one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polyrnerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA