posted on 2015-04-17, 00:00authored byNicole
A. Leal, Hyo-Joong Kim, Shuichi Hoshika, Myong-Jung Kim, Matthew A. Carrigan, Steven A. Benner
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 polymerases 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.