title: Transcription, reverse transcription, and analysis of RNA containing artificial genetic components.
abstract: 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.
authors.affiliation: †Foundation for Applied Molecular Evolution (FfAME), 720 SW Second Avenue, Suite 201, Gainesville, Florida 32601, United States.|‡Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Leal
authors.affiliation: †Foundation for Applied Molecular Evolution (FfAME), 720 SW Second Avenue, Suite 201, Gainesville, Florida 32601, United States.|‡Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Hyo-Joong
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: †Foundation for Applied Molecular Evolution (FfAME), 720 SW Second Avenue, Suite 201, Gainesville, Florida 32601, United States.|‡Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hoshika
authors.affiliation: †Foundation for Applied Molecular Evolution (FfAME), 720 SW Second Avenue, Suite 201, Gainesville, Florida 32601, United States.|‡Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Myong-Jung
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: §Department of Natural Sciences, Santa Fe College, 3000 NW 83rd Street L209, Gainesville, Florida 32606, United States.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Carrigan
authors.affiliation: †Foundation for Applied Molecular Evolution (FfAME), 720 SW Second Avenue, Suite 201, Gainesville, Florida 32601, United States.|‡Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D004247:DNA / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D012313:RNA / Q000096:biosynthesis*; D048348:Reverse Transcription; D058615:Synthetic Biology / Q000379:methods; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: T7 RNA polymerase; artificially expanded genetic information systems; reverse transcriptase; synthetic biology; transcription