Expanding the limits of the second genetic code with ribozymes

Area of Science:

• Biochemistry
• Synthetic Biology
• Molecular Biology

Background:

• Genetic code reprogramming enables the synthesis of novel bio-based products by incorporating noncanonical monomers into polypeptides.
• Flexizymes, which are transfer RNA (tRNA) synthetase-like ribozymes, have been utilized to broaden the range of chemical substrates for ribosome-directed polymerization.
• Developing design rules for flexizyme-catalyzed acylation is crucial for the scalable and rational expansion of genetic code reprogramming.

Purpose of the Study:

• To systematically synthesize and evaluate a diverse set of noncanonical monomers for their compatibility with flexizyme-mediated tRNA acylation and subsequent in vitro translation.
• To establish design rules for flexizyme-catalyzed acylation based on the electronic and steric properties of various monomer scaffolds.
• To demonstrate the utility of the derived design rules in predicting and incorporating novel monomers, including those enabling orthogonal bioconjugation.

Main Methods:

• Systematic synthesis of 37 substrates across four diverse chemical scaffolds: phenylalanine, benzoic acid, heteroaromatic, and aliphatic monomers.
• Evaluation of substrate acylation onto tRNA and subsequent incorporation into peptides using in vitro translation systems.
• Analysis of structure-activity relationships to derive design rules for flexizyme-mediated acylation.

Main Results:

• 32 out of 37 synthesized substrates were successfully acylated onto tRNA and incorporated into peptides.
• Established design rules for flexizyme-catalyzed acylation based on systematic substrate synthesis and in vitro translation data.
• Successfully predicted and incorporated 6 additional monomers, including one that facilitates N-terminal aldehyde incorporation for orthogonal bioconjugation.

Conclusions:

• The developed design rules provide a framework for the rational and scalable expansion of the genetic code through flexizyme-mediated incorporation of noncanonical monomers.
• This expanded synthetic capability enables the creation of peptides with unique functionalities, such as orthogonal bioconjugation.
• The study advances the field of synthetic biology by providing tools and strategies for engineering novel bio-based products beyond natural limitations.