Special considerations for on-demand design, generation, and evolution of macromolecules that rely on non-canonical/unnatural building blocks.

Engineering Biology

Special considerations for on-demand design, generation, and evolution of macromolecules that rely on non-canonical/unnatural building blocks.

Current State-of-the-Art

While DNA, RNA, and proteins containing natural building blocks are readily synthesized using natural biological machinery and the rules of templated biosynthesis, DNA, RNA, and proteins containing modified or unnatural building blocks, including ones that recapitulate post-translational modifications, are difficult to access. Only certain unnatural building blocks are available, only a few distinct unnatural building blocks can be used simultaneously, and the length of fully unnatural polymers that can be produced is extremely low compared to natural counterparts. Overcoming these bottlenecks will lead to new biomolecules with expanded functions stemming from the diversity of non-canonical and unnatural building blocks that could become available to synthetic biology.

The design, generation, and evolution of macromolecules containing unnatural building blocks relies on the achievement of the same capabilities as the production of wholly-natural macromolecules. This Goal reflects the special considerations necessary for the utilization of unnatural building blocks.

Breakthrough Capabilities & Milestones

PCR, reverse transcription, cellular replication, and transcription of fully unnatural nucleotide-containing genes of up to 400 base pairs.

At this length, unnatural aptamer and aptazyme polymers could be regularly evolved and engineered.

Identification of “missing” functionality or functionalities in A-T-G-C base pairs.

Improved in vitro manipulation of unnatural nucleic acids.

Expansion of unnatural nucleotide toolkit.

Bottleneck/Challenge: At present, transcription and translation of DNA containing unnatural base pairs has been achieved only in the context of a single specialized unnatural base pair and only in E. coli.

Bottleneck/Challenge: Success in this endeavor required extensive optimization.

Potential Solution: Begin to explore alternative (previously explored) unnatural base pairs in the context of the optimized conditions, especially ones that do not perturb the double helical structure of DNA and can be incorporated in any sequence context.

Biosynthesis of unnatural nucleotides.

Organisms capable of full replication, maintenance, and transcription of a plasmid or artificial chromosome made up entirely of unnatural bases.

Expanded genetic code systems for translation of >100-amino acid proteins containing fully-unnatural amino acids, and proteins with at least four distinct unnatural amino acid building blocks.

Create proteins that are capable of gaining new, therapeutically-useful activities through unnatural amino acids.

Efficient biosynthesis of proteins containing three or more distinct unnatural amino acid building blocks.

Bottleneck/Challenge: Not enough free codons to hijack for unnatural amino acid building blocks.

Potential Solution: Genomically-recoded strains that free up redundant codons, orthogonal ribosomes that can utilize special tRNAs for special messages, or orthogonal organellar genetic codes.

Bottleneck/Challenge: Not enough mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs for unnatural building block incorporation.

Potential Solution: Design or scalable evolution of new mutually orthogonal sets of aaRS/tRNA pairs for genomically-recoded organisms or for orthogonal ribosomes; hijacking of organellar genetic codes and associated aaRS/tRNA pairs.