The engineering of semi-synthetic organisms seemed to have stalled. Yes, they could be equipped with unnatural base pairs (UBPs), and DNA sequences containing these UBPs could be stably maintained and transcribed, leading to the production of proteins containing noncanonical amino acids (ncAAs). But the production of proteins was disappointingly slow. In this sense, semi-synthetic organisms were just less efficient than natural organisms.
To help semi-synthetic organisms pick up the pace, scientists based at Scripps Research decided to optimize them by exploring a variety of deoxy- and ribonucleotide analogues. The researchers tested different combinations of UBPs in Escherichia coli and observed which ones were replicated most efficiently and produced the highest levels of a protein.
Some of the synthetic base pairs had been tested before, whereas others were new variations. The team then used these optimized base pairs to demonstrate, for the first time, a semi-synthetic organism that could make a protein containing multiple ncAAs. And the production of this protein was gratifyingly efficient.
Detailed findings appeared in a paper (“Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism”) that appeared recently in the Journal of the American Chemical Society. The paper describes how the Scripps Research team, which was led by Floyd E. Romesberg, PhD, professor at Scripps Research, observed similarities and differences between the ways DNA and RNA polymerases recognized the unnatural nucleotides.
“Remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs,” the article’s authors wrote. “The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval.”
All of Earth’s natural life forms store information using a four-letter genetic code consisting of the nucleotides deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and deoxythymidine (dT). Within the DNA double helix, dA pairs with dT, and dG with dC, to form the “rungs” of the DNA ladder.
Recently, researchers have made synthetic nucleotides that can pair up with each other. When they placed these unnatural nucleotides into genes, bacteria could replicate the DNA and convert the sequences into RNA and then proteins that contained unconventional amino acids.
Extending this work, the Scripps Research team were motivated to elucidate the structure–activity relationships (SARS) governing the templating of transcription. For example, the scientists conducted an in vivo SAR analysis of unnatural ribonucleoside triphosphates.
“From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation,” the authors of the current study concluded. “The optimized semi-synthetic organism is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.”