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

Abstract

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.

In Vivo Assembly and Expression of DNA Containing Non‐canonical Bases in the Yeast Saccharomyces cerevisiae

“…This might be due to the use of the forcing PCR condition that lowered the DNA polymerase fidelity towards nucleoside misincorporation (e. g., error prone Taq polymerase with elevated concentration of Mg 2+ ions was used) [22] . These results align with our previous reports, [11,12] where 7‐deazaA was identified as the most mutagenic nucleotide among the other modifications. Despite the incorporation of errors in the synthetic gene, the fully functional protein was expressed in a eukaryotic organism from DZA genetic material even when all four nucleotides were substituted to their non‐canonical counterparts.…”

“…Previously, we demonstrated that partially or fully substituted DZA fragments conferring resistance to antibiotic or fluorescent reporter gene could accurately transfer genetic information in E. coli [11,12] . Here, we were questioning whether eukaryotic organisms could read and process the information from the synthetic DZAs as well.…”

“…Furthermore, 237‐bp long, fully modified XNA genes (denoted as DZA) comprised of the four non‐canonical nucleotides – 7‐deaza‐adenine pairing with 5‐chlorouracil and 7‐deaza‐guanine or deoxyinosine pairing with 5‐fluorocytosine or 5‐bromocytosine – encoding R67 dihydrofolate reductase (R67 DHFR) have been synthesized and successfully expressed in E. coli [11] . The DZA‐comprising genes were further explored to encode 777‐bp‐long red fluorescent protein in bacteria [12] . Interesting, in artificial cells comprising E. coli gene expression machinery, 4′‐thioG‐containing DNA template can be transcribed into 4′‐thioC RNA and subsequently expressed in functional green fluorescent protein [13] .…”

“…[11] The DZA-comprising genes were further explored to encode 777-bp-long red fluorescent protein in bacteria. [12] Interesting, in artificial cells comprising E. coli gene expression machinery, 4'-thioG-containing DNA template can be transcribed into 4'-thioC RNA and subsequently expressed in functional green fluorescent protein. [13] Apart from the nucleobase and sugar modified genes, the phosphodiester linkage modifications were studied as genetic templates.…”

Traceless enzymatic synthesis of monodispersed hypermodified oligodeoxyribonucleotide polymers from RNA templates

“…In the past, there were only several scattered examples 9,10 of polymerase synthesis of DNA containing all-four modified bases and one set of four small base-modified nucleotides (not decorated by functional groups) was used for in vivo chemical morphing of bacteria. 11…”