Expandiendo y reprogramando el código genético

Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chemical and physical properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes.

Protein translation uses transfer RNAs (tRNAs), which are aminoacylated with their cognate amino acids by aminoacyl-tRNA synthetase enzymes, to read triplet codons in messenger RNAs (mRNAs) via base pairing interactions between the mRNA codon and the anticodon of the tRNA. The ribosome facilitates both the sequential decoding of triplet codons on mRNAs by cognate tRNAs, and the polymerization of the corresponding amino acids into a polypeptide (Fig. 1). The near-universal genetic code defines how 64 triplet codons are decoded in a template-directed manner on the ribosome to synthesize proteins with complex three-dimensional structures. The code is read by an evolutionarily conserved translational machinery and, with the notable exceptions of the atypical amino acids selenocysteine and pyrrolysine, all proteins are synthesized from a limited set of building blocks: the 20 canonical amino acids.

Figure 1: The natural translational machinery performs encoded amino acid polymerization.

An aminoacyl-tRNA synthetase (aaRS) aminoacylates its cognate tRNA with a specific amino acid (blue circle). The aminoacylated tRNA is decoded on the ribosome in response to a cognate codon in the mRNA during translational elongation, leading to the addition of an amino acid to the growing polymer. Iterative decoding of aminoacylated tRNAs leads to the synthesis of an amino acid polymer with a defined sequence.

Over the past two decades, approaches have been developed to expand the genetic code, enabling the co-translational and site-specific incorporation of diverse noncanonical amino acids (ncAAs) into proteins synthesized in cells. These approaches extend strategies for the stoichiometric, chemical aminoacylation of tRNAs—for use in in vitro studies or (via injection) in oocytes—to diverse in vivo settings, using catalytically and specifically aminoacylated tRNAs, and complement other strategies for the synthesis of modified proteins.

Figure 2: Genetic code expansion for ncAA incorporation into proteins in vivo and its use for creating attenuated viruses.

ncAAs and viral genes containing amber (TAG) codons are introduced into cells containing the orthogonal aminoacyl-tRNA synthetase (O-aaRS) and orthogonal tRNA. The orthogonal aaRS transfers the ncAA onto the tRNA, and the aminoacylated tRNA is used to decode UAG codons in the viral mRNA. Virus particles containing UAG codons in their packaged genomes are produced from these cells. Immunization with this ‘replication-incompetent virus’ leads to protection in animal models.

The site-specific incorporation of diverse chemistries into proteins has led to a wave of powerful, transformative new ways to control, evolve and understand biological functions and to provide new strategies for medicines. Ongoing efforts are testing our ability to radically engineer the translational machinery, and to recode entire genomes, with the goal of reprogramming organisms to synthesize noncanonical biopolymers. Here I focus on key developments in in vivo code expansion over the past three years and on emerging strategies that may contribute to in vivocode reprogramming in the future; more details on earlier in vivo work can be found in previous reviews.

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