scholarly journals Optimization of Expanded Genetic Codes via Genetic Algorithms

2018 ◽  
Author(s):  
Maísa de Carvalho Silva ◽  
Lariza Laura De Oliveira ◽  
Renato Tinós
Keyword(s):  
Amino Acids ◽  
Genetic Algorithms ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Genetically Modified Organisms ◽  
Fitness Function ◽  
Unnatural Amino Acid ◽  
Standard Genetic Code ◽  
Genetic Codes

In the last decades, researchers have proposed the use of genetically modified organisms that utilize unnatural amino acids, i.e., amino acids other than the 20 amino acids encoded in the standard genetic code. Unnatural amino acids have been incorporated into genetically engineered organisms for the development of new drugs, fuels and chemicals. When new amino acids are incorporated, it is necessary to modify the standard genetic code. Expanded genetic codes have been created without considering the robustness of the code. The objective of this work is the use of genetic algorithms (GAs) for the optimization of expanded genetic codes. The GA indicates which codons of the standard genetic code should be used to encode a new unnatural amino acid. The fitness function has two terms; one for robustness of the new code and another that takes into account the frequency of use of amino acids. Experiments show that, by controlling the weighting between the two terms, it is possible to obtain more or less amino acid substitutions at the same time that the robustness is minimized.

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 On the Importance of Asymmetry in the Phenotypic Expression of the Genetic Code upon the Molecular Evolution of Proteins

Symmetry ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 997
Author(s):  
Marco V. José ◽  
Gabriel S. Zamudio
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Phenotypic Expression ◽  
Standard Genetic Code ◽  
Specific Amino Acid ◽  
Trna Synthetases ◽  
Probability Of Occurrence ◽  
Genetic Codes ◽  
Evolution Of Proteins

The standard genetic code (SGC) is a mapping between the 64 possible arrangements of the four RNA nucleotides (C, A, U, G) into triplets or codons, where 61 codons are assigned to a specific amino acid and the other three are stop codons for terminating protein synthesis. Aminoacyl-tRNA synthetases (aaRSs) are responsible for implementing the SGC by specifically amino-acylating only its cognate transfer RNA (tRNA), thereby linking an amino acid with its corresponding anticodon triplets. tRNAs molecules bind each codon with its anticodon. To understand the meaning of symmetrical/asymmetrical properties of the SGC, we designed synthetic genetic codes with known symmetries and with the same degeneracy of the SGC. We determined their impact on the substitution rates for each amino acid under a neutral model of protein evolution. We prove that the phenotypic graphs of the SGC for codons and anticodons for all the possible arrangements of nucleotides are asymmetric and the amino acids do not form orbits. In the symmetrical synthetic codes, the amino acids are grouped according to their codonicity, this is the number of triplets encoding a given amino acid. Both the SGC and symmetrical synthetic codes exhibit a probability of occurrence of the amino acids proportional to their degeneracy. Unlike the SGC, the synthetic codes display a constant probability of occurrence of the amino acid according to their codonicity. The asymmetry of the phenotypic graphs of codons and anticodons of the SGC, has important implications on the evolutionary processes of proteins.

scholarly journals High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

2013 ◽  
Vol 80 (5) ◽  
pp. 1718-1725 ◽  
Author(s):  
Masaomi Minaba ◽  
Yusuke Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Expression System ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Translational Efficiency ◽  
Site Specific ◽  
Content Type

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

scholarly journals Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

2007 ◽  
Vol 283 (3) ◽  
pp. 1525-1533 ◽  
Author(s):  
Shixin Ye ◽  
Caroline Köhrer ◽  
Thomas Huber ◽  
Manija Kazmi ◽  
Pallavi Sachdev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
G Protein Coupled Receptors ◽  
Calcium Flux ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

scholarly journals Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 409
Author(s):  
Tamara L. Hendrickson ◽  
Whitney N. Wood ◽  
Udumbara M. Rathnayake
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Chemical Reactivity ◽  
Trna Synthetase ◽  
Amino Acid Side Chain ◽  
Standard Genetic Code ◽  
Last Universal Common Ancestor ◽  
Trna Synthetases ◽  
Universal Common Ancestor

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.

Incorporation of Unnatural Amino Acids into Cytochrome c3 and Specific Viologen Binding to the Unnatural Amino Acid

ChemBioChem ◽  
2006 ◽  
Vol 7 (12) ◽  
pp. 1853-1855 ◽  
Author(s):  
Shin Iida ◽  
Noriyuki Asakura ◽  
Kenji Tabata ◽  
Ichiro Okura ◽  
Toshiaki Kamachi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
Cytochrome C3

Many alternative and theoretical genetic codes are more robust to amino acid replacements than the standard genetic code

2019 ◽  
Vol 464 ◽  
pp. 21-32 ◽  
Author(s):  
Paweł Błażej ◽  
Małgorzata Wnętrzak ◽  
Dorota Mackiewicz ◽  
Przemysław Gagat ◽  
Paweł Mackiewicz
Keyword(s):  
Amino Acid ◽  
Genetic Code ◽  
Standard Genetic Code ◽  
Genetic Codes

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.

scholarly journals Sterilization of drug-resistant influenza virus through genetic interference: use of unnatural amino acid-engineered virions

2021 ◽  
Author(s):  
Xuesheng Wu ◽  
Zhetao Zheng ◽  
Hongmin Chen ◽  
Haishuang Lin ◽  
Yuelin Yang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Influenza A ◽  
Antiviral Agent ◽  
Unnatural Amino Acid ◽  
Drug Resistant ◽  
Genetic Code Expansion

AbstractThe frequent emergence of drug resistance during the treatment of influenza A virus (IAV) infections highlights a need for effective antiviral countermeasures. Here, we present an antiviral method that utilizes unnatural amino acid-engineered drug-resistant (UAA-DR) virus. The engineered virus is generated through genetic code expansion to combat emerging drug-resistant viruses. The UAA-DR virus has unnatural amino acids incorporated into its drug-resistant protein and its polymerase complex for replication control. The engineered virus can undergo genomic segment reassortment with normal virus and produce sterilized progenies due to artificial amber codons in the viral genome. We validate in vitro that UAA-DR can provide a broad-spectrum antiviral strategy for several H1N1 strains, different DR-IAV strains, multidrug-resistant (MDR) strains, and even antigenically distant influenza strains (e.g., H3N2). Moreover, a minimum dose of neuraminidase (NA) inhibitors for influenza virus can further enhance the sterilizing effect when combating inhibitor-resistant strains, partly due to the promoted superinfection of unnatural amino acid-modified virus in cellular and animal models. We also exploited the engineered virus to achieve adjustable efficacy after external UAA administration, for mitigating DR virus infection on transgenic mice harboring the pair, and to have substantial elements of the genetic code expansion technology, which further demonstrated the safety and feasibility of the strategy. We anticipate that the use of the UAA-engineered DR virion, which is a novel antiviral agent, could be extended to combat emerging drug-resistant influenza virus and other segmented RNA viruses.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Gregory M. Olenginski ◽  
Juliana Piacentini ◽  
Darcy R. Harris ◽  
Nicolette A. Runko ◽  
Brianna M. Papoutsis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Unnatural Amino Acid ◽  
Green Fluorescent ◽  
Absorbance Band ◽  
Protein Constructs

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

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