The Network of Replication, Transcription, and Reverse Transcription of a Synthetic Genetic Cassette

Synthetic nucleic acids, with four non-canonical nucleobases, can function as genetic materials. A comprehensive analysis of PCR amplification, transcription, reverse transcription, and cloning was done to screen for alternative genetic monomers. A small library of six modified nucleobases was selected: the modified 2'-deoxyribonucleoside (dZTPs) and ribonucleoside (rZTPs) triphosphates of 7-deaza-adenine, 5-chlorouracil, 7-deaza-guanine or inosine together with 5-fluorocytosine or 5-bromocytosine. The fragments composed of one to four modified nucleotides (denoted as DZA) have been successfully recognized and transcribed to natural or modified RNA (denoted as RZA) by T7 RNA polymerase. The fully modified RZA fragment could be reverse transcribed and then amplified in the presence of various dZTPs. Noticeably, modified fragments could function as genetic templates in vivo by encoding the 678 base pair gene of a fluorescent protein in bacteria. These results demonstrate the existence of a fully simulated genetic circuit that uses synthetic materials.