Genetic Code Expansion Technology Research Articles

Insights into the dynamics of the Ca2+ release-activated Ca2+ channel pore-forming complex Orai1.

An important calcium (Ca2+) entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel, which controls a series of downstream signaling events such as gene transcription, secretion and proliferation. It is composed of a Ca2+ sensor in the endoplasmic reticulum (ER), the stromal interaction molecule (STIM), and the Ca2+ ion channel Orai in the plasma membrane (PM). Their activation is initiated by receptor-ligand binding at the PM, which triggers a signaling cascade within the cell that ultimately causes store depletion. The decrease in ER-luminal Ca2+ is sensed by STIM1, which undergoes structural rearrangements that lead to coupling with Orai1 and its activation. In this review, we highlight the current understanding of the Orai1 pore opening mechanism. In this context, we also point out the questions that remain unanswered and how these can be addressed by the currently emerging genetic code expansion (GCE) technology. GCE enables the incorporation of non-canonical amino acids with novel properties, such as light-sensitivity, and has the potential to provide novel insights into the structure/function relationship of CRAC channels at a single amino acid level in the living cell.

TRNA engineering strategies for genetic code expansion.

The advancement of genetic code expansion (GCE) technology is attributed to the establishment of specific aminoacyl-tRNA synthetase/tRNA pairs. While earlier improvements mainly focused on aminoacyl-tRNA synthetases, recent studies have highlighted the importance of optimizing tRNA sequences to enhance both unnatural amino acid incorporation efficiency and orthogonality. Given the crucial role of tRNAs in the translation process and their substantial impact on overall GCE efficiency, ongoing efforts are dedicated to the development of tRNA engineering techniques. This review explores diverse tRNA engineering approaches and provides illustrative examples in the context of GCE, offering insights into the user-friendly implementation of GCE technology.

Affinity-Directed Site-Specific Protein Labeling and Its Application to Antibody-Drug Conjugates.

Chemically modified proteins have diverse applications; however, conventional chemo-selective methods often yield heterogeneously labeled products. To address this limitation, site-specific protein labeling holds significant potential, driving extensive research in this area. Nevertheless, site-specific modification of native proteins remains challenging owing to the complexity of their functional groups. Therefore, a method for site-selective labeling of intact proteins is aimed to design. In this study, a novel approach to traceless affinity-directed intact protein labeling is established, which leverages small binding proteins and genetic code expansion technology. By applying this method, a site-specific antibody labeling with a drug, which leads to the production of highly effective antibody-drug conjugates specifically targeting breast cancer cell lines is achieved. This approach enables traceless conjugation of intact target proteins, which is a critical advantage in pharmaceutical applications. Furthermore, small helical binding proteins can be easily engineered for various target proteins, thereby expanding their potential applications in diverse fields. This innovative approach represents a significant advancement in site-specific modification of native proteins, including antibodies. It also bears immense potential for facilitating the development of therapeutic agents for various diseases.

Applications of genetic code expansion technology in eukaryotes.

Unnatural amino acids (UAAs) have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins. In eukaryotes, genetic code expansion (GCE) enables the incorporation of UAAs and facilitates posttranscriptional modification (PTM), which is not feasible in prokaryotic systems. GCE is also a powerful tool for cell or animal imaging, the monitoring of protein interactions in target cells, drug development, and switch regulation. Therefore, there is keen interest in utilizing GCE in eukaryotic systems. This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.

Applications of genetic code expansion and photosensitive UAAs in studying membrane proteins.

Membrane proteins are the targets for most drugs and play essential roles in many life activities in organisms. In recent years, unnatural amino acids (UAAs) encoded by genetic code expansion (GCE) technology have been widely used, which endow proteins with different biochemical properties. A class of photosensitive UAAs has been widely used to study protein structure and function. Combined with photochemical control with high temporal and spatial resolution, these UAAs have shown broad applicability to solve the problems of natural ion channels and receptor biology. This review will focus on several application examples of light-controlled methods to integrate GCE technology to study membrane protein function in recent years. We will summarize the typical research methods utilizing some photosensitive UAAs to provide common strategies and further new ideas for studying protein function and advancing biological processes.

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light.

Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.

Shedding light on ORAI1 channel with genetic code expansion

Genetic code expansion technology has been widely applied to control protein activity and biological systems by taking advantage of an amber stop codon suppressor tRNA and orthogonal aminoacyl-tRNA synthetase pair. With this chemical biology approach, Maltan et al. incorporated photocrosslinking unnatural amino acids (UAAs) into the transmembrane domains of ORAI1 to enable UV light-inducible calcium influx across the plasma membrane, mechanistic interrogation of the calcium release-activated calcium (CRAC) channel at the single amino acid level, and remote control of downstream calcium-modulated signaling in mammalian cells.

Functional analysis of protein post-translational modifications using genetic codon expansion.

Post-translational modifications (PTMs) of proteins not only exponentially increase the diversity of proteoforms, but also contribute to dynamically modulating the localization, stability, activity, and interaction of proteins. Understanding the biological consequences and functions of specific PTMs has been challenging for many reasons, including the dynamic nature of many PTMs and the technical limitations to access homogenously modified proteins. The genetic code expansion technology has emerged to provide unique approaches for studying PTMs. Through site-specific incorporation of unnatural amino acids (UAAs) bearing PTMs or their mimics into proteins, genetic code expansion allows the generation of homogenous proteins with site-specific modifications and atomic resolution both in vitro and in vivo. With this technology, various PTMs and mimics have been precisely introduced into proteins. In this review, we summarize the UAAs and approaches that have been recently developed to site-specifically install PTMs and their mimics into proteins for functional studies of PTMs.

Optimized genetic code expansion technology for time-dependent induction of adhesion GPCR-ligand engagement.

The introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair enables site-specific incorporation of unnatural amino acids (uAAs) with functionalized side chains into proteins of interest. Genetic Code Expansion (GCE) via amber codon suppression confers functionalities to proteins but can also be used to temporally control the incorporation of genetically encoded elements into proteins. Here, we report an optimized GCE system (GCEXpress) for efficient and fast uAA incorporation. We demonstrate that GCEXpress can be used to efficiently alter the subcellular localization of proteins within living cells. We show that click labeling can resolve co-labeling problems of intercellular adhesive protein complexes. We apply this strategy to study the adhesion G protein-coupled receptor (aGPCR) ADGRE5/CD97 and its ligand CD55/DAF that play central roles in immune functions and oncological processes. Furthermore, we use GCEXpress to analyze the time course of ADGRE5-CD55 ligation and replenishment of mature receptor-ligand complexes. Supported by fluorescence recovery after photobleaching (FRAP) experiments our results show that ADGRE5 and CD55 form stable intercellular contacts that may support transmission of mechanical forces onto ADGRE5 in a ligand-dependent manner. We conclude that GCE in combination with biophysical measurements can be a useful approach to analyze the adhesive, mechanical and signaling properties of aGPCRs and their ligand interactions. This article is protected by copyright. All rights reserved.

Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222.

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.

Studying Reversible Protein Post-translational Modification through Co-translational Modification.

Understanding the post-translational modifications of targeted proteins is of great significance for manipulating the physiological processes of eukaryotes. Chemical biology tools have been used to investigate the biological roles of those post-translational modifications at particular sites, especially genetic code expansion technology, which can also be combined with the concept of synthetic biology to generate a genetically modified organism with a synthetic auxotroph for co-translational modification components. In this concept, we will introduce applications, limitations, and perspectives of genetic code expansion technology for studying post-translational modification based on recent progresses. Future perspectives of genetically modified organisms also will be discussed in regard to the application of post-translational modification research.

Site-specific vibrational spectroscopy with non-canonical amino acids containing frequency-resolved labels.

Unnatural protein side-chains with frequencies within the “transparent window” spectral region (∼1800 - 2700 cm−1), where no native protein vibrations are found, can single out specific residues in vibrational spectra. These moieties contain most often triple bonds, like nitriles (R-C≡N), which can be conveniently attached to proteins in the form of genetically encoded non-canonical amino acids (ncAA) e.g. 4-cyanophenylalanine (CNF). However, the full potential of ncAA as vibrational reporters has not been exploited yet, particularly for Raman spectroscopy. Here we show how CNF residues introduced at multiple positions of the bacterial transcription factor EL222 provide a detailed picture of local changes along the photocycle. Time-resolved infrared spectra in the CNF absorption region disclose two additional kinetic events not sensed by the native probes, one occurring before flavin chromophore relaxation and the other after protein backbone relaxation. In addition, we incorporate an ncAA carrying a diacetylene (R-C≡C-C≡CH) via genetic code expansion technology into EL222 variants. The conjugated diyne features an intense Raman signal which is sensitive to the microenvironment around the triple bonds. We then measure light-induced conformational changes in the flavoprotein EL222 with single-residue precision. Our results suggest that vibrational spectroscopy assisted by ncAA can reveal protein structural dynamics residue-by-residue.

Photoregulation of IL-24/IL-20R2 complex formation using a light-responsive non-canonical amino acid.

Human Interleukin 24 (IL-24) is an immunomodulatory cytokine that represents an important target for treatment of autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with its cell membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers many exciting possibilities in basic research and may have also potential applications in therapy. In particular, control of protein-protein interactions by light has recently found increasing interest. As a proof-of-concept, we developed a strategy based on genetic code expansion technology to photo-control the binding between IL-24 and one of its receptors, IL-20R2. Both these proteins are engineered versions of the native proteins that feature high stability and can be recombinantly expressed in E. coli at adequate yields. Introduction of the photocaged non-canonical amino acid ortho-nitrobenzyl-tyrosine (ONBY) at selected positions of IL-20R2 resulted in severe impairment of heterocomplex assembly as determined by microscale thermophoresis. Irradiation of ONBY-substituted IL-20R2 with UV light (365 nm) reconstitutes the canonical tyrosines and subsequently restores the ability of IL-20R2 to recognize IL-24. We envision that photocontrollable IL-20R2 may be useful for the spatiotemporal regulation of signaling pathways in which IL-24 is involved.

Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for theAnalysis of Deubiquitinase Activities.

The genetic code expansion technology enables the genetic encoding of fluorescent noncanonical amino acids (ncAAs) for site-specific fluorescent labeling of proteins. These co-translational and internal fluorescent tags have been harnessed to establish genetically encoded Förster resonance energy transfer (FRET) probes for studying protein structural changes and interactions. Here, we describe the protocols for site-specific incorporation of an aminocoumarin-derived fluorescent ncAA into proteins in E. coli and preparation of a fluorescent ncAA-based FRET probe for assaying theactivities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases.

A novel viral vaccine platform based on engineered transfer RNA

ABSTRACT In recent years, an increasing number of emerging and remerging virus outbreaks have occurred and the rapid development of vaccines against these viruses has been crucial. Controlling the replication of premature termination codon (PTC)-containing viruses is a promising approach to generate live but replication-defective viruses that can be used for potent vaccines. Here, we used anticodon-engineered transfer RNAs (ACE-tRNAs) as powerful precision switches to control the replication of PTC-containing viruses. We showed that ACE-tRNAs display higher potency of reading through PTCs than genetic code expansion (GCE) technology. Interestingly, ACE-tRNA has a site preference that may influence its read-through efficacy. We further attempted to use ACE-tRNAs as a novel viral vaccine platform. Using a human immunodeficiency virus type 1 (HIV-1) pseudotyped virus as an RNA virus model, we found that ACE-tRNAs display high potency for read-through viral PTCs and precisely control their production. Pseudorabies virus (PRV), a herpesvirus, was used as a DNA virus model. We found that ACE-tRNAs display high potency for reading through viral PTCs and precisely controlling PTC-containing virus replication. In addition, PTC-engineered PRV completely attenuated and lost virulence in mice in vivo, and immunization with PRV containing a PTC elicited a robust immune response and provided complete protection against wild-type PRV challenge. Overall, replication-controllable PTC-containing viruses based on ACE-tRNAs provide a new strategy to rapidly attenuate virus infection and prime robust immune responses. This technology can be used as a platform for rapidly developing viral vaccines in the future.

Engineering enzyme activity using an expanded amino acid alphabet.

Enzyme design and engineering strategies are typically constrained by the limited size of nature's genetic alphabet, comprised of only 20 canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, with hundreds of structurally diverse ncAAs now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering. ncAAs have been used to probe complex biological mechanisms, augment enzyme function and, most ambitiously, embed new catalytic mechanisms into protein active sites that would be challenging to access within the constraints of nature's genetic code. We predict that the studies reviewed in this article, along with further advances in genetic code expansion technology, will establish ncAA incorporation as an increasingly important tool for biocatalysis in the coming years.

Systematic dissection of key factors governing recombination outcomes by GCE-SCRaMbLE

With the completion of Sc2.0 chromosomes, synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) becomes more critical for in-depth investigation of fundamental biological questions and screening of industrially valuable characteristics. Further applications, however, are hindered due to the lack of facile and tight regulation of the SCRaMbLE process, and limited understanding of key factors that may affect the rearrangement outcomes. Here we propose an approach to precisely regulate SCRaMbLE recombination in a dose-dependent manner using genetic code expansion (GCE) technology with low basal activity. By systematically analyzing 1380 derived strains and six yeast pools subjected to GCE-SCRaMbLE, we find that Cre enzyme abundance, genome ploidy and chromosome conformation play key roles in recombination frequencies and determine the SCRaMbLE outcomes. With these insights, the GCE-SCRaMbLE system will serve as a powerful tool in the future exploitation and optimization of the Sc2.0-related technologies.

Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid.

Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of O-methyltyrosine (OMeY). Specifically, we endowed organisms with a marformycins biosynthetic pathway-derived methyltransferase that efficiently converts tyrosine to OMeY in the presence of the co-factor S-adenosylmethionine. The resulting cells can produce and site-specifically incorporate OMeY into proteins at much higher levels than cells exogenously fed OMeY. To understand the structural basis for the substrate selectivity of the transferase, we solved the X-ray crystal structures of the ligand-free and tyrosine-bound enzymes. Most importantly, we have extended this OMeY biosynthetic system to both mammalian cells and the zebrafish model to enhance the utility of genetic code expansion. The creation of autonomous eukaryotes using a 21st amino acid will make genetic code expansion technology more applicable to multicellular organisms, providing valuable vertebrate models for biological and biomedical research.

Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation

Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

A Light-Activatable Photocaged Variant of the Ultra-High Affinity ALFA-Tag Nanobody.

Nanobodies against short linear peptide‐epitopes are widely used to detect and bind proteins of interest (POI) in fusion constructs. Engineered nanobodies that can be controlled by light have found very recent attention for various extra‐ and intracellular applications. We here report the design of a photocaged variant of the ultra‐high affinity ALFA‐tag nanobody, also termed ALFA‐tag photobody. ortho‐Nitrobenzyl tyrosine was incorporated into the paratope region of the nanobody by genetic code expansion technology and resulted in a ≥9,200 to 100,000‐fold impairment of the binding affinity. Irradiation with light (365 nm) leads to decaging and reconstitutes the native nanobody. We show the light‐dependent binding of the ALFA‐tag photobody to HeLa cells presenting the ALFA‐tag. The generation of the first photobody directed against a short peptide epitope underlines the generality of our photobody design concept. We envision that this photobody will be useful for the spatiotemporal control of proteins in many applications using cultured cells.