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.
Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the ...efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (
C. elegans
and
D. melanogaster
) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.
Reprogramming the genetic code de la Torre, Daniel; Chin, Jason W
Nature reviews. Genetics,
03/2021, Letnik:
22, Številka:
3
Journal Article
Recenzirano
The encoded biosynthesis of proteins provides the ultimate paradigm for high-fidelity synthesis of long polymers of defined sequence and composition, but it is limited to polymerizing the canonical ...amino acids. Recent advances have built on genetic code expansion - which commonly permits the cellular incorporation of one type of non-canonical amino acid into a protein - to enable the encoded incorporation of several distinct non-canonical amino acids. Developments include strategies to read quadruplet codons, use non-natural DNA base pairs, synthesize completely recoded genomes and create orthogonal translational components with reprogrammed specificities. These advances may enable the genetically encoded synthesis of non-canonical biopolymers and provide a platform for transforming the discovery and evolution of new materials and therapeutics.
Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA ...synthetase/
tRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/Mm
tRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/Mm
tRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/
tRNA
pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/
tRNA pairs that are mutually orthogonal to the MmPylRS/Mm
tRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/
tRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.
Designer amino acids, beyond the canonical 20 that are normally used by cells, can now be site-specifically encoded into proteins in cells and organisms. This is achieved using 'orthogonal' ...aminoacyl-tRNA synthetase-tRNA pairs that direct amino acid incorporation in response to an amber stop codon (UAG) placed in a gene of interest. Using this approach, it is now possible to study biology in vitro and in vivo with an increased level of molecular precision. This has allowed new biological insights into protein conformational changes, protein interactions, elementary processes in signal transduction and the role of post-translational modifications.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
8.
Expanding the Genetic Code of an Animal Greiss, Sebastian; Chin, Jason W
Journal of the American Chemical Society,
09/2011, Letnik:
133, Številka:
36
Journal Article
Recenzirano
Odprti dostop
Genetic code expansion, for the site-specific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code ...of a multicellular animal, the nematode Caenorhabditis elegans.
Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires ...the discovery of multiple orthogonal aminoacyl-transfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/
tRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/
tRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and
tRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with
tRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/
tRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/
tRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.
The efficient, site-specific introduction of unnatural amino acids into proteins in mammalian cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. ...Addressing this challenge will allow the synthesis of modified recombinant proteins and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in cells. The efficiency of unnatural amino acid incorporation in response to the amber stop codon (UAG) in mammalian cells is commonly considered to be low. Here we demonstrate that tRNA levels can be limiting for unnatural amino acid incorporation efficiency, and we develop an optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system, with optimized tRNA expression for mammalian cells. In addition, we engineer eRF1, that normally terminates translation on all three stop codons, to provide a substantial increase in unnatural amino acid incorporation in response to the UAG codon without increasing readthrough of other stop codons. By combining the optimized pyrrolysyl-tRNA synthetase/tRNACUA expression system and an engineered eRF1, we increase the yield of protein bearing unnatural amino acids at a single site 17- to 20-fold. Using the optimized system, we produce proteins containing unnatural amino acids with comparable yields to a protein produced from a gene that does not contain a UAG stop codon. Moreover, the optimized system increases the yield of protein, incorporating an unnatural amino acid at three sites, from unmeasurably low levels up to 43% of a no amber stop control. Our approach may enable the efficient production of site-specifically modified therapeutic proteins, and the quantitative replacement of targeted cellular proteins with versions bearing unnatural amino acids that allow imaging or synthetic regulation of protein function.
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