Writing code for synthetic life

In the high school my chemistry teacher used to tell us that chemists do not only study the nature, but they also invent their own subject of study. The more I learn about biology, the more I feel that biologists move in the same direction, creating the new field of synthetic biology.

Recent report on the expansion of DNA alphabet by two letters grossly overshadowed not that press-release-friendly development in the synthetic biology of RNA. But there are quite some interesting things going on in the latter field that deserve as much attention.

In general, alphabet expansion is of no use if one can’t make sensible words of new letters. In the cellular context that means efficient transcription and translation of the genetic code. Tinkering with ‘pre-built’ cellular machinery would likely cause a mess of unpredictable outcomes due to the sheer complexity of regulatory feedback loops. Hence, great deal of efforts is applied to the development of orthogonal translation machinery.

Jason Chin group is one of the leaders in the area. Back in 2005 the group reported the development of orthogonal (O-)ribosomes and O-mRNA, which could produce proteins without interfering with natural cellular homeostasis. To achieve this, they mutated short sequence fragment upstream of AUG start codon, which is responsible for mRNA recognition by  rRNA (i.e. Shine-Dalgarno sequence). Thus, normal ribosomes didn’t recognize O-mRNA, O-ribosomes didn’t recognize normal mRNA, and O-ribosomes recognized O-mRNA. Based on similar O-ribosome construct five years later Chin et al. could incorporate unnatural amino acids into the polypeptide chain of calmoduline by using quadruplet codons (instead of natural triplets) and engineered tRNAse synthetase. This allowed theoretical expansion of the genetic code to 200 unnatural amino acids.

The work of Chin group is based on systematic evolution of 16S rRNA (small ribosomal subunit) of E. coli. This subunit is responsible for recognition of mRNA. But fully functional ribosomes need also large 23S subunit to translate the message. To exclude the possibility of hijacking of natural large subunits by orthogonal rRNAs, one can tether both subunits covalently. In 2015, Jason Chin lab successfully achieved that but was outstripped by Alexander Mankin lab, whose paper in Nature was published a month earlier.

Both groups used circular permutation approach for the design of synthetic ribosomes. That means they connected the 3′ and 5′ ends of wild-type 23S, which are spatially close to each other, and cut the rRNA string in one of the apical loops identified from the secondary structure of 23S. Then 16S sequence was incorporated between new 3′ and 5′ ends, so two subunits could be transcribed as a single message. Although Mankin lab did comprehensive screen for allowed permutations and Chin lab designed their connection based only on known structure of ribosome, both groups ended up connecting two rRNA subunits via helix H101 of 23S and h44 of 16S. The linkers were different, however. Chin lab used J5/J5a region from the Tetrahymena group I self-splicing intron, sequence that can adopt linear (extended) or U-turn confrormation. Mankin lab linker design was a bit less sophisticated, they just screened a bunch of oligo(A) linkers and found that 8-9 nucleotides is the optimal length.

16S-23S_Mankin
(left) Secondary structure of WT rRNAs 23S and 16S; (right) secondary structure of Ribo-T from Mankin lab. Source

To introduce orthogonality in their ribosome design, Mankin lab borrowed the approach from the seminal Chin’s paper. Hence, they obtained oRibo-T1 construct. This was further improved by bacteria: unplanned spontaneous mutation in the PL promoter yielded more robust E. coli strain than originally transformed with oRibo-T1.

Both groups then nicely demonstrated independence of orthogonal ribosomes from endogenous subunits by introducing functional mutations (Chin lab) in combination with mechanism-specific antibiotic treatment (Mankin lab). In general, the Nature paper is more mechanism-based, while the Angewandte paper focuses more on whole-cell effects of stapled O-ribosomes.

Spurred by newly emerged competition, the independent development of synthetic ribosomes by two different groups promises some exciting advances in the field in the nearest future. Best luck to the postdocs in both groups!

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Author: Slava Bernat

I did my PhD in medicinal chemistry/chemical biology of G protein-coupled receptors and then explored some chemical biology of non-coding RNA as a postdoc. Currently I'm working in a small biotech company in San-Francisco Bay area as a research chemist. I'm writing about science, which catches my attention in rss feed reader and some random thoughts or tutorials.

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