Genetic Code Engineering by Natural and Unnatural Base Pair Systems for the Site-Specific Incorporation of Non-Standard Amino Acids Into Proteins

Kimoto, Michiko; Hirao, Ichiro

doi:10.3389/fmolb.2022.851646

Cited by 17 publications

(11 citation statements)

References 121 publications

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“…Introduction of an unnatural base (UB) pair that does not interact with A•T(U) or C•G could expand the genetic alphabet and theoretically create hundreds of new codons available for incorporation of npMs (Figure 18d). 448 The major challenge faced by this field is that replication of DNA, transcription of RNA, and translation of proteins are all built upon natural nucleotides; thus, each of these processes must be made compatible with UBs. 449 While each of these processes come with their own inherent challenges and have been discussed in detail in other reviews (see refs 448−451), this section focuses on UBs which have been used in translation.…”

Section: Artificial Division Of Codon Boxes By Sensementioning

confidence: 99%

“…A newer avenue for engineered, npMcontaining proteins is for therapeutic purposes, with several currently in clinical trials. 448 A common strategy is to genetically encode an npM with a bioorthogonal handle at an amber stop codon and then conjugate on a polyethylene glycol (PEG)-based moiety. 478 For example, the biopharmaceutical company Ambrx utilized this strategy to express an anti-HER2 antibody with pAcPhe at position 121 and conjugate on a nonhydrolyzable hydroxylamine-based PEG linker with a potent synthetic drug.…”

Section: In Vivo Expression Of Proteins and Peptides Containing Non-p...mentioning

confidence: 99%

“…Engineered tRNAs have been used for the in vivo incorporation of over 150 npMs into proteins for a wide variety of functions, such as for site-selective labeling, spectroscopic probes, post-translational modifications, and cross-linking (reviewed in refs , , , and ). A newer avenue for engineered, npM-containing proteins is for therapeutic purposes, with several currently in clinical trials . A common strategy is to genetically encode an npM with a bioorthogonal handle at an amber stop codon and then conjugate on a polyethylene glycol (PEG)-based moiety .…”

Section: Applications Of Polypeptides Containing Ribosomally Translat...mentioning

confidence: 99%

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Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

Sigal,

Matsumoto,

Beattie

et al. 2024

Chem. Rev.

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Ribosome-dependent protein biosynthesis is an essential cellular process mediated by transfer RNAs (tRNAs). Generally, ribosomally synthesized proteins are limited to the 22 proteinogenic amino acids (pAAs: 20 L-α-amino acids present in the standard genetic code, selenocysteine, and pyrrolysine). However, engineering tRNAs for the ribosomal incorporation of non-proteinogenic monomers (npMs) as building blocks has led to the creation of unique polypeptides with broad applications in cellular biology, material science, spectroscopy, and pharmaceuticals. Ribosomal polymerization of these engineered polypeptides presents a variety of challenges for biochemists, as translation efficiency and fidelity is often insufficient when employing npMs. In this Review, we will focus on the methodologies for engineering tRNAs to overcome these issues and explore recent advances both in vitro and in vivo. These efforts include increasing orthogonality, recruiting essential translation factors, and creation of expanded genetic codes. After our review on the biochemical optimizations of tRNAs, we provide examples of their use in genetic code manipulation, with a focus on the in vitro discovery of bioactive macrocyclic peptides containing npMs. Finally, an analysis of the current state of tRNA engineering is presented, along with existing challenges and future perspectives for the field.

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Section: Artificial Division Of Codon Boxes By Sensementioning

confidence: 99%

Section: In Vivo Expression Of Proteins and Peptides Containing Non-p...mentioning

confidence: 99%

Section: Applications Of Polypeptides Containing Ribosomally Translat...mentioning

confidence: 99%

See 1 more Smart Citation

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

Sigal,

Matsumoto,

Beattie

et al. 2024

Chem. Rev.

View full text Add to dashboard Cite

show abstract

“…Last but not least, the specific complementary base pairings of A-T/U and G-C, based on hydrogen bonds in living organisms, have been exploited to build hybrid organic/inorganic nanostructures (Kimoto & Hirao, 2022). For example, a DNA-modified phages could bind to hundreds of complementary DNA-coated Au NPs through base-pairing, allowing for the accurate detection of antigens in solution based on the optical changes caused by the aggregation of Au (Lee et al, 2012).…”

Section: Biological Reconstructionsmentioning

confidence: 99%

Living virus‐based nanohybrids for biomedical applications

Jin,

Mao

2023

WIREs Nanomed Nanobiotechnol

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Living viruses characterized by distinctive biological functions including specific targeting, gene invasion, immune modulation, and so forth have been receiving intensive attention from researchers worldwide owing to their promising potential for producing numerous theranostic modalities against diverse pathological conditions. Nevertheless, concerns during applications, such as rapid immune clearance, altering immune activation modes, insufficient gene transduction efficiency, and so forth, highlight the crucial issues of excessive therapeutic doses and the associated biosafety risks. To address these concerns, synthetic nanomaterials featuring unique physical/chemical properties are frequently exploited as efficient drug delivery vehicles or treatments in biomedical domains. By constant endeavor, researchers nowadays can create adaptable living virus‐based nanohybrids (LVN) that not only overcome the limitations of virotherapy, but also combine the benefits of natural substances and nanotechnology to produce novel and promising therapeutic and diagnostic agents. In this review, we discuss the fundamental physiochemical properties of the viruses, and briefly outline the basic construction methodologies of LVN. We then emphasize their distinct diagnostic and therapeutic performances for various diseases. Furthermore, we survey the foreseeable challenges and future perspectives in this interdisciplinary area to offer insights.This article is categorized under: Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies

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“…Finally, one can resort to various types of genetic code expansion techniques [121][122][123], in this work only stop codon suppression (SCS) was used and is explained in detail. 6 The SCS approach exploits the usage of stop codons for the incorporation of ncAAs.…”

Section: Protein Labeling For Spectroscopymentioning

confidence: 99%

Protein vibrational energy transfer: new sensors and application in an enzyme active site

Löffler

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Experiments on Vibrational Energy Transfer (VET) in proteins contribute to our understanding of fundamental biological processes such as allostery, dissipation of excess energy, and possibly enzymatic catalysis. While these processes have been studied for a long time, many questions remain unanswered. The aim of this work was to expand the application of existing spectroscopic techniques to investigate VET, seeking tailored solutions for the diversity of proteins and amino acid environments. Additionally, new target proteins were to be established to broaden the spectrum of VET experiments towards the role of VET and low-frequency protein modes (LFMs). To test their suitability as VET sensors, the non-canonical amino acids (ncAAs) Azidoalanine (N3Ala), azido-L-Homoalanine (Aha), p-azido-Phenylalanine (N3Phe), p-cyano-Phenylalanine (CNPhe), and 4-cyano-Tryptophan (CNTrp) were coupled to the VET donor β-(1-azulenyl)-L-Alanine (AzAla) in dipeptides. Their spectral properties were compared using FTIR and VET spectra in H2O, dimethyl sulfoxide, and tetrahydrofuran. The solvent strongly influences the measured VET signals, which can be explained by the direct interaction of the solvent with the dipeptides. Additionally, the peak time within the subgroups of azide and nitrile sensors increased with the size of the side chain, indicating the dependence between peak time and the distance between VET donor and sensor. When incorporated into a protein, solvent interactions are less dominant. Therefore, Aha, N3Phe, and CNPhe were additionally incorporated at two different positions in the PDZ protein domain and investigated. Due to Fermi resonances, signals from azide sensors are challenging to predict, unlike those of the nitrile sensors. Overall, the experiments showed that nitrile groups can serve well as VET sensors, as their lower extinction coefficient is compensated for by a narrower bandwidth. This expands the number of potential target proteins, and sensor incorporation can be less disruptive at various protein locations. Since the VET donor AzAla can inject the energy of a photon into a protein as vibrational energy at a specific location, it can also be used for the targeted excitation of LFMs. If these modes are involved in an enzymatic reaction, a direct influence on activity is expected. This hypothesis has long existed but has not been definitively verified. Some studies have found evidence for the involvement of LFMs in formate dehydrogenase (FDH) catalysis. Therefore, FDH was chosen for the investigation of LFMs in enzymes. This specific system additionally allows the use of a natural VET sensor: it forms a stable complex with NAD+ and N3-, an excellent IR marker. Thus, it provided the opportunity to test low-molecular-weight non-covalent ligands as VET sensors. After ensuring sufficient AzAla supply through the internal establishment of an enzymatic synthesis, AzAla could be incorporated at various positions in FDH. Despite spectral overlap between free and bound N3-, the latter could be identified by its narrower FWHM. For some variants, no binding could be observed. Circular dichroism spectra showed that these variants structurally deviate slightly from other variants and the wild type (WT). VET could be observed over 22 Å from two regions of the protein to the N3- bound in the active center, at protein concentrations of below 2 mM. Unbound N3- did not generate signals, allowing it to be added in excess ensuring the saturation of the protein in VET experiments. The activity of FDH WT and four AzAla mutants was investigated under substrate saturation without and with AzAla excitation. In these experiments, a slight reduction in activity under illumination was observed, even for the WT, who is not expected to interact with the excitation light. So far, a difference in sample temperature cannot be excluded as the cause for this decline. The presented experiments with FDH illustrate the potential of low-molecular-weight ligands as VET sensors, with N3- being particularly attractive due to its simple structure (preventing Fermi resonances) and its high extinction coefficient. Its use can add many metalloproteins as potential targets for VET experiments and allows investigation without a VET sensor ncAA. Additionally, initial experiments were conducted to measure light-dependent FDH activity. By specifically exciting protein LFMs, this project could contribute in the future to answering longstanding questions about the extraordinary catalytic efficiency of enzymes.

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Genetic Code Engineering by Natural and Unnatural Base Pair Systems for the Site-Specific Incorporation of Non-Standard Amino Acids Into Proteins

Cited by 17 publications

References 121 publications

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers

Living virus‐based nanohybrids for biomedical applications

Protein vibrational energy transfer: new sensors and application in an enzyme active site

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