scholarly journals Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion, in the World of tRNA Modifications

2022 ◽  
Vol 23 (2) ◽  
pp. 938
Author(s):  
Olubodun Michael Lateef ◽  
Michael Olawale Akintubosun ◽  
Olamide Tosin Olaoba ◽  
Sunday Ocholi Samson ◽  
Malgorzata Adamczyk
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Protein Design ◽  
Side Chains ◽  
Vast Number ◽  
Trna Modifications ◽  
Wide Range ◽  
Challenges And Opportunities ◽  
Code Extension ◽  
Genetic Code Expansion

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.

scholarly journals Pyrrolysyl-tRNA-Synthetase: Methanogenese und Gencode-Erweiterung

BIOspektrum ◽  
2021 ◽  
Vol 27 (6) ◽  
pp. 616-619
Author(s):  
Nikolaj Georg Koch ◽  
Nediljko Budisa
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Methanogenic Archaea ◽  
Trna Synthetase ◽  
Side Chains ◽  
Site Specific ◽  
Genetic Code Expansion ◽  
The Way

AbstractPyrrolysyl-tRNA synthetase (PylRS) is an enzyme of some methanogenic Archaea for the natural incorporation of pyrrolysine into proteins. The discovery of PylRS as a natural tool for genetic code expansion paved the way for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins, with versatile side chains useful in biotechnology. Almost 20 years after the discovery, we describe the journey which led to three distinct classes of PylRSs with unique ncAA recognitions.

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

scholarly journals Some theoretical aspects of reprogramming the standard genetic code

2020 ◽  
Author(s):  
Kuba Nowak ◽  
Paweł Błażej ◽  
Małgorzata Wnetrzak ◽  
Dorota Mackiewicz ◽  
Paweł Mackiewicz
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Dna Sequences ◽  
Theoretical Perspective ◽  
Optimal Number ◽  
Optimal Size ◽  
Coding System ◽  
Standard Genetic Code ◽  
Nucleotide Mutation ◽  
Code Extension

1AbstractReprogramming of the standard genetic code in order to include non-canonical amino acids (ncAAs) opens a new perspective in medicine, industry and biotechnology. There are several methods of engineering the code, which allow us for storing new genetic information in DNA sequences and transmitting it into the protein world. Here, we investigate the problem of optimal genetic code extension from theoretical perspective. We assume that the new coding system should encode both canonical and new ncAAs using 64 classical codons. What is more, the extended genetic code should be robust to point nucleotide mutation and minimize the possibility of reversion from new to old information. In order to do so, we follow graph theory to study the properties of optimal codon sets, which can encode 20 canonical amino acids and stop coding signal. Finally, we describe the set of vacant codons that could be assigned to new amino acids. Moreover, we discuss the optimal number of the newly incorporated ncAAs and also the optimal size of codon blocks that are assigned to ncAAs.

Rational design of a function-based selection method for genetically encoding acylated lysine derivatives

2019 ◽  
Vol 17 (25) ◽  
pp. 6127-6130
Author(s):  
Hui Miao ◽  
Chenguang Yu ◽  
Anzhi Yao ◽  
Weimin Xuan
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Rational Design ◽  
Selection Method ◽  
Genetic Code Expansion

Genetic code expansion depends on the directed evolution of aaRS to recognize non-canonical amino acids. Herein, we reported a function-based method that enables rapidly evolving aaRS for acylated lysine derivatives.

scholarly journals The Role of Orthogonality in Genetic Code Expansion

Life ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 58 ◽  
Author(s):  
Pol Arranz-Gibert ◽  
Jaymin R. Patel ◽  
Farren J. Isaacs
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Genetic Code ◽  
Cross Reactivity ◽  
Elongation Factors ◽  
Translational Machinery ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Genetic Code Expansion

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

The Synthesis of a Cubane-Substituted Dipeptide

2012 ◽  
Vol 65 (6) ◽  
pp. 690 ◽  
Author(s):  
Quentin I. Churches ◽  
Roger J. Mulder ◽  
Jonathan M. White ◽  
John Tsanaktsidis ◽  
Peter J. Duggan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Sensitivity ◽  
Pure Form ◽  
Bioactive Molecules ◽  
Side Chains ◽  
Cyclic Hydrocarbon ◽  
Wide Range ◽  
Effective Synthesis ◽  
Dipeptide Derivative

Amino acids and peptides bearing cyclic hydrocarbon side-chains are of interest in the development of a wide range of bioactive molecules. The preparation of an amino acid and a dipeptide derivative bearing an unfunctionalised cubane substituent is described. Attempts to prepare a cubylalanine derivative via the corresponding dehydroalanine were unsuccessful due to the high sensitivity of this vinyl cubane compound. Conversely, the addition of cubyllithium to a (RS)-glyoxylate sulfinimine led to an effective synthesis of a cubylglycine derivative and a cubane-substituted dipeptide in diastereomerically pure form.

scholarly journals Basic principles of the genetic code extension

2019 ◽  
Author(s):  
Paweł Błażej ◽  
Małgorzata Wnetrzak ◽  
Dorota Mackiewicz ◽  
Paweł Mackiewicz
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Point Mutations ◽  
Coding System ◽  
Base Pairs ◽  
Induced Subgraphs ◽  
Single Nucleotide ◽  
Basic Principles ◽  
Code Extension ◽  
Incremental Addition

AbstractCompounds including non-canonical amino acids or other artificially designed molecules can find a lot of applications in medicine, industry and biotechnology. They can be produced thanks to the modification or extension of the standard genetic code (SGC). Such peptides or proteins including the non-canonical amino acids can be constantly delivered in a stable way by organisms with the customized genetic code. Among several methods of engineering the code, using non-canonical base pairs is especially promising, because it enables generating many new codons, which can be used to encode any new amino acid. Since even one pair of new bases can extend the SGC up to 216 codons generated by six-letter nucleotide alphabet, the extension of the SGC can be achieved in many ways. Here, we proposed a stepwise procedure of the SGC extension with one pair of non-canonical bases to minimize the consequences of point mutations. We reported relationships between codons in the framework of graph theory. All 216 codons were represented as nodes of the graph, whereas its edges were induced by all possible single nucleotide mutations occurring between codons. Therefore, every set of canonical and newly added codons induces a specific subgraph. We characterized the properties of the induced subgraphs generated by selected sets of codons. Thanks to that, we were able to describe a procedure for incremental addition of the set of meaningful codons up to the full coding system consisting of three pairs of bases. The procedure of gradual extension of the SGC makes the whole system robust to changing genetic information due to mutations and is compatible with the views assuming that codons and amino acids were added successively to the primordial SGC, which evolved to minimize harmful consequences of mutations or mistranslations of encoded proteins.

scholarly journals Genetically Encoded Libraries of Nonstandard Peptides

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Takashi Kawakami ◽  
Hiroshi Murakami
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Posttranslational Modifications ◽  
Biological Properties ◽  
Peptide Libraries ◽  
Universal Genetic Code ◽  
Nonsense Codons ◽  
Genetic Code Expansion ◽  
Diagnostic Applications ◽  
Selection Of

The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.

scholarly journals Evolving Bacterial Fitness with an Expanded Genetic Code

2017 ◽  
Author(s):  
Drew S. Tack ◽  
Austin C. Cole ◽  
R. Shroff ◽  
B.R. Morrow ◽  
Andrew D. Ellington
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acid ◽  
Noncanonical Amino Acids ◽  
Bacterial Fitness ◽  
Genetic Code Expansion ◽  
Encoding Strategy ◽  
Translation Systems ◽  
Expanded Genetic Code

AbstractEvolution has for the most part used the canonical 20 amino acids of the natural genetic code to construct proteins. While several theories regarding the evolution of the genetic code have been proposed, experimental exploration of these theories has largely been restricted to phylogenetic and computational modeling. The development of orthogonal translation systems has allowed noncanonical amino acids to be inserted at will into proteins. We have taken advantage of these advances to evolve bacteria to accommodate a 21 amino acid genetic code in which the amber codon ambiguously encodes either 3-nitro-L-tyrosine or stop. Such an ambiguous encoding strategy recapitulates numerous models for genetic code expansion, and we find that evolved lineages first accommodate the unnatural amino acid, and then begin to evolve on a neutral landscape where stop codons begin to appear within genes. The resultant lines represent transitional intermediates on the way to the fixation of a functional 21 amino acid code.

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