Implications of Codon Usage, tRNA Gene Redundancy and tRNA Gene Clustering in Experimental Models of Mistranslation.

Site-specific incorporation of photo-responsive unnatural amino acids (UAAs) into proteins via genetic code expansion offers a powerful approach to control and study protein function in biological systems. However, existing UAAs are all sensitive to UV or near-UV light, and no visible-light-responsive UAAs have been reported, limiting our ability to regulate multiple proteins simultaneously. Here, we present the genetic encoding of a green-light-activatable lysine derivative, SCouK, for sequential photocontrol of protein activities in live cells. SCouK, containing a photolabile thiocoumarin moiety at the N ε-amino group of lysine, can be genetically encoded into proteins in bacterial and mammalian cells. We show that site-specifically incorporated SCouK can be photoactivated across a broad wavelength range, from UV to green light, to restore the functions of EGFP and luciferase. Notably, SCouK is highly efficiently photodecaged by green light centered at 520 nm within 30 seconds, marking it as the first visible-light-responsive lysine derivative with the longest single-photon activation wavelength among genetically encoded photolabile UAAs. Additionally, we showcase the general capability of SCouK for the optical control of different kinases and temporal control and interrogation of the cGAS-STING pathway in live cells. Moreover, by combing SCouK with a UV-light-activatable tyrosine derivative, we achieve, for the first time, sequential photoactivation of two distinct UAA-modified proteins within a single live-cell sample. Overall, the unique features of SCouK, including site-specific incorporation, green-light-responsiveness, orthogonal activation wavelengths, high decaging efficiency, and general applicability, demonstrate its great potential to non-invasively and precisely manipulate proteins in complex living systems for functional studies and therapeutic applications.

Purification-free immobilization of dopamine D2 receptor and glucocorticoid receptor by alkyne-azide cycloaddition for affinity chromatography-based drug screening.

Single-molecule tracking (SMT) is a powerful tool for real-time studies of protein interactions in living cells. Dye-labelled SNAP-tag and HaloTag self-labelling proteins have simplified SMT significantly, due to their superior photophysical properties compared to fluorescent proteins. However, due to their size, fusion of these tags to a protein of interest often results in loss of protein function. We introduce FLORENCE - a universal labelling method for SMT, based on genetic code expansion (GCE). We overcome significant caveats related to re-coded strains, vectors, and dyes and report successful tracking of site-specifically intracellularly labelled proteins in genomically re-coded E. coli. Our findings establish a robust in vivo protein-labelling strategy, expanding the capabilities of SMT as a method to study the dynamics of proteins in living cells. Moreover, we observe that the strain-promoted azide-alkyne click-chemistry reaction occurs as fast as 30 min in live E. coli cells and can be used as a robust labelling reaction.

Tyrosine kinases control a wide range of cell signaling pathways that are central to human physiology, and they are dysregulated in a variety of human diseases, most notably cancers. Our understanding of tyrosine kinase biology hinges upon a clear delineation of their protein substrates. Thus, much effort has been invested into defining the substrate specificities of tyrosine kinases, which is partly driven by recognition of the amino acid sequences surrounding the phospho-acceptor tyrosine residues. Numerous methods have been developed to profile tyrosine kinase sequence recognition, and these approaches have collectively demonstrated that different tyrosine kinases have distinct substrate sequence preferences. Here, we describe one such method that combines bacterial peptide display and deep sequencing to study tyrosine kinase substrate preferences. Our approach enables rapid measurement of relative phosphorylation efficiencies for thousands of peptides simultaneously. The genetically-encoded nature of the peptide libraries used with this method allows for facile and cheap construction of libraries tailored to answer a variety questions. Notably, our approach is compatible with genetic code expansion via Amber codon suppression, which allows for the construction and screening of libraries containing non-canonical amino acids. Importantly, results from this assay correlate strongly with quantitative measurements of enzyme kinetics, they corroborate previously reported tyrosine kinase substrate preferences, and they can reveal new insights into tyrosine kinase substrate specificity.

Abstract Waveform design plays a crucial role in modern radar systems, where minimizing range sidelobes is essential for improving detection accuracy and reducing false alarms. Conventional approaches such as Phase Coding (PC), Linear Frequency Modulation (LFM), and Nonlinear Frequency Modulation (NLFM) often suffer from high sidelobe levels and poor robustness under Doppler shifts, limiting their practical effectiveness. To overcome these drawbacks, this paper proposes a Quantum Genetic Algorithm-based Complementary Amplitude Coding (CAC-QGA) framework for optimized waveform design. The integration of complementary amplitude coding with QGA ensures effective exploration of the solution space, leading to a balanced minimization of Peak Sidelobe Ratio (PSLR), Integrated Sidelobe Ratio (ISLR), and Generalized Integrated Sidelobe Level (GISL). By combining simulated annealing-based elitist selection and three genetic engineering strategies Dominant Chromosome Identification, Directed Mutation, and Gene Injection this framework enhances both convergence speed and robustness against local optima. The algorithm further achieves improved computational efficiency through adaptive population updating and quantum rotation-based encoding, reducing convergence time compared to conventional metaheuristics. Simulation results confirm that CAC-QGA achieves a PSLR of 21.1 dB, an ISLR of 0.35 dB, and a GISL of –19.1 dB, outperforming existing optimization-based waveform designs. Compared to state-of-the-art algorithms, CAC-QGA reduces execution time by an average of over 27% and memory usage by 18%, demonstrating its superiority in both accuracy and resource utilization. Additionally, the method exhibits faster convergence and increased robustness under Doppler frequency variations, proving its effectiveness in practical, dynamic radar environments. These findings highlight CAC-QGA as a high-efficiency and reliable optimization framework for advanced radar waveform design.

The construction of a circular code through a biological process, particularly a primitive one in the absence of the protein world, has remained an open problem since the discovery of a maximal [Formula: see text] self-complementary trinucleotide circular code in genes in 1996 (Arquès and Michel, 1996). Circular codes are defined by their ability to recover the correct reading frame of genes at any position. While a class of 216 such trinucleotide codes has been identified, the KL method (Koch and Lehman, 1997), based on nucleotide probability products, generates only a restricted subclass of 88 [Formula: see text]-codes (Lacan and Michel, 2001). Revisiting this probabilistic framework 25 years later, we demonstrate that various classes of dinucleotide circular codes can be generated using a nucleotide probability product model (called Construction 2). We introduce the concept of transitive dinucleotide codes and prove new theorems characterizing their circularity and comma-free properties. Using codon usage from bacteria, archaea, and eukaryotes, 2 "universal" maximal dinucleotide circular codes are observed: [Formula: see text] in the codon site [Formula: see text] and [Formula: see text] in the codon site [Formula: see text] which can be deduced from [Formula: see text] by 1-letter cyclical permutation [Formula: see text] or identically by reversing permutation [Formula: see text]. Unexpectedly, we then show that, under the independence assumption, the dinucleotide code [Formula: see text] through Construction 2 from nucleotide frequencies in the codon sites 1 and 2, is a maximal dinucleotide circular code and is equal to the observed dinucleotide code: [Formula: see text]. These findings support a theoretical model in which dinucleotide circular codes may have originated from statistical properties of primitive nucleotide distributions, providing insights into the possible emergence of the genetic code.

Statement of the problem. Historically, the concepts of “soul” and “spirit” of a city have existed for thousands of years. They have entered the culture of nations and peoples, into traditions, myths, legends and heroic epics. They are used for self-identification by citizens of individual states or the population of individual local territories. Although no one gives clear answers to the question of what is the “soul” and what is the “spirit” in the physical space of a specific city. Attempts to formulate answers based on religious ideas about the “soul” and “spirit” of a city generally lead away from the answer, since theological doctrines assert that the soul and spirit are not material, and therefore there is no need to look for them in the material context of settlements. They are not there. Other documents from the past describe how the soul and spirit of the city were worshipped, how mutual aid treaties were made with them. Their locations were known. They were guarded by people, and they guarded people. At the same time, modern research of processes related to the concept of “soul” and “spirit” of the city, carried out by academic and alternative scientists, gradually leads to the understanding that the areas of existence of these concepts are in the primary structures of the quantum reality of our world. And even deeper. They belong to the ranges of energies whose properties go beyond the boundaries of space and time. The soul and spirit of the city can already be registered by scientific instruments. Moreover, there have always been, are and will be indirect, physically visible, numerous indicators of these “beyond” phenomena in the visible ranges of human existence. They are “non-instrument devices” for detecting the spirit, soul and genetic code in the development of any city. Thus, two non-material components – spirit and soul - are manifested through the material component of the city – the genetic code of its planning structure. Or, conversely, a material detector shows the presence of the non-material “anatomy” of the city. The concept of “genetic code of the city planning structure” arose in the seventies of the twentieth century. However, in various explanations and terms it was recorded in ancient times. And, nevertheless, in the interpretation of this concept there are still many different interpretations and ambiguities. It is time to describe these three phenomena based on the use of modern physical approaches. The aim of the article is to reveal the physical meaning and features of the interaction of the concepts of “spirit”, “soul” and “genetic code” of the city.

Pharmacogenomics (PGx) is a scientific field that aims to understand how an individual's genetic code regulates drug metabolism and response. The response to many anesthetic drugs varies widely among patients due to many factors including, but not limited to, age, gender, and comorbidities. However, PGx contributes to this variability, particularly regarding adverse drug reactions. This review explores the influence of PGx on five commonly used induction agents in anesthesia: propofol, midazolam, ketamine, etomidate, and thiopental. Propofol metabolism is significantly affected by polymorphisms in CYP2B6, CYP2C9, and UGT1A9, influencing both efficacy and toxicity. Midazolam's PGx is mainly mediated by variations in CYP3A4, CYP3A5, and UDP-glucuronosyltransferase enzymes, with implications for sedation depth and drug clearance. Ketamine response is modulated by polymorphisms in metabolic enzymes (e.g. CYP2B6), as well as neurobiological targets such as brain-derived neurotrophic factor and gamma-aminobutyric acid (GABA) receptors, particularly in psychiatric applications. Etomidate shows less studied but emerging PGx associations, including single-nucleotide polymorphisms in GABA receptor subunits and metabolic enzymes, which may affect both sedative depth and cardiovascular stability. Thiopental is a rapid-acting metabolite whose effect stems from GABA-A receptor potentiation; no studies have yet identified specific genetic polymorphisms influencing its action. Overall, PGx provides a promising avenue for tailoring anesthetic management to improve patient safety and outcomes. However, clinical integration remains limited due to practical and infrastructural barriers. This review highlights the potential and current limitations of pharmacogenomic-guided anesthesia, underscoring its relevance in the era of precision medicine.

Oral delivery of therapeutic proteins remains a formidable challenge. Although engineered microbes have emerged as promising platforms for localized drug synthesis in the gut, their functional capacity has been restricted to the 20 canonical amino acids, limiting the chemical diversity of biologic payloads. Here, we demonstrate that integrating genetic code expansion (GCE) into bacterial therapy overcomes this fundamental constraint. We engineered the probiotic Escherichia coli Nissle 1917 (EcN) to incorporate the noncanonical amino acid fluorosulfate-l-tyrosine (FSY), enabling in situ secretion of a site-specifically modified covalent anti-IL-23 nanobody exhibiting picomolar binding potency (5.9 pM). Oral administration of this engineered EcN strain, followed by FSY supplementation, significantly ameliorated colitis in a murine model. This approach thereby establishes a versatile and generalizable platform that substantially expands the functional scope and therapeutic potential of live biotherapeutics.

DNA codon mutations involving Stop signals or the amino acid cysteine can be especially damaging. The former can break protein sequences or add extraneous amino acids. The latter can add or subtract disulfide bonds crucial in protein folding. We present a hypothetical scenario where Stop codons were present early in the evolution of the genetic code; and minimizing catastrophic mutations for code networks affected all subsequent amino acid/codon pairings. Predicted features of this "Catastrophic Mutation Minimization Hypothesis" (CMMH) are that: (1) Cysteine is mutationally adjacent to Stop, isolating a contiguous codon 'neighborhood' with high potential for catastrophe. (2) The sequence of amino acid additions order determines codon assignments through minimizing network-wide mutation costs. Overall, codon locations for 16 of the 20 amino acids in the genetic code are consistent with the CMMH, as are multiple other predictions. We propose an antecedent genetic code consisted of 16 doublet codons specifying 13-14 amino acids. Two variations of these networks are less susceptible to catastrophic mutations than 88.2-97.5% of randomly generated ones. Unlike some previous hypotheses, CMMH does not require the total replacement or rearrangement of amino acids at codons, with its disruptive potential for protein synthesis. Finally, the composition of this ancestral doublet genetic code has all the modern code's utility: amino acids from four chemical types; start and stop signals; metal-binding ability; disulfide bridging for creating protein shapes; and possible epigenetic gene regulation. Thus, the modern code likely evolutionarily fine-tuned antecedent capabilities, rather than significantly increasing competence for making complex proteins.

Transfer RNAs (tRNAs) ensure accurate decoding of the genetic code. However, mutations in tRNAs can lead to misincorporation of an amino acid that differs from the genetic message in a process known as mistranslation. As mistranslating tRNAs modify how the genetic message is decoded, they have potential as therapeutic tools for diseases caused by nonsense and missense mutations. Despite this, they also produce proteome-wide mismade proteins, which can disrupt proteostasis. To better understand the impact of mistranslating tRNA variants, we profile the proteome and phosphoproteome of yeast expressing three different mistranslating tRNAs. While the overall impacts were similar, the extent of growth defects and proteome changes varied with the substitution type. Although the global impacts were modest, mistranslation influenced key cellular processes, including proteostasis, cell cycle, and translation. These findings highlight the need to consider cellular consequences when developing mistranslating tRNAs for therapeutic applications.

As outstanding representatives of traditional cotton textile techniques in Hubei Province, the Hong'an Dabu and Zaoyang coarse cloth making techniques carry the genetic code of Jingchu farming civilization. Taking the two "Dabu" techniques as research objects, this paper adopts literature research, field investigation and comparative analysis methods to conduct a systematic comparison from five dimensions: historical origin, technological process, core techniques, artistic expression and inheritance mode. The study finds that Hong'an Dabu technique is characterized by complicated processes and infiltration of revolutionary culture, while Zaoyang coarse cloth is distinguished by streamlined procedures and diverse patterns. Both share the technical foundation of Hubei's cotton textile industry and form unique features due to regional cultural differences. Finally, the paper proposes an inheritance path of "technical mutual learning + cultural empowerment + design innovation", providing theoretical support for the modern transformation of Hubei's traditional textile techniques.

RNA serves as both a genetic messenger and a functional non-coding molecule, with its activity relying on interactions with diverse proteins. Characterizing RNA-protein interactions remains challenging, particularly for dynamic or low-abundance complexes. Traditional crosslinking methods, such as UV-254nm irradiation and formaldehyde fixation, suffer from low efficiency, poor specificity, and broad reactivity, limiting their utility for high-resolution interactome mapping. Recent advances in genetic code expansion (GCE) and unnatural amino acid (UAA) incorporation now enable chemoselective, site-specific crosslinking chemistries that circumvent these limitations. This review highlights classes of crosslinkable UAAs, including benzophenone-, diazirine-, and aryl azide-based moieties, which have been used for photo-induced covalent capture of RNA-protein interactions. Next-generation modalities, such as acetophenone derivatives, halogenated UAAs, bifunctional UAAs, acyl silanes, diaryl nitrones, and photoactivatable systems further expand the toolkit for photocrosslinking and bioorthogonal labelling. Beyond UV-based approaches, latent bioreactive UAAs and proximity-induced chemistries exploit electrophile-nucleophile substitution and masked acylating agents to enable covalent capture under physiological conditions. Together, these strategies provide unprecedented control over reactivity, temporal resolution and site specificity, advancing RNA-targeted proteomics. Continued innovation in UAA-based chemistry promise to transform how RNA-protein complexes are interrogated and manipulated with molecular precision.

A novel strategy based on genetic code expansion combined with click chemistry for the simultaneous visualization of distinct posttranslational modification (PTM) states of a single protein within living cells. As a model, it is focused on threonine 41 (T41) of β‐catenin, a regulatory hotspot implicated in epithelial cancers and known to be phosphorylated, O‐GlcNAcylated, or left unmodified. Using site‐specific incorporation of the unnatural phenylselenocysteine, a β‐catenin‐EGFP fusion protein is engineered allowing selective installation of a S‐GlcNAc moiety via oxidative elimination and thiol‐Michael ligation. Additional β‐catenin variants, phosphomimetic T41E‐mCherry and wild‐type‐blue fluorescent protein fusions, are produced to represent other PTM states. All constructs are successfully introduced into Hep3B and HeLa cells by lipofection or TAT‐mediated transduction. Fluorescence microscopy revealed distinct subcellular localization profiles for each PTM form. Notably, the S‐GlcNAcylated β‐catenin exhibited enhanced resistance to proteasomal degradation, consistent with known roles of O‐GlcNAcylation in protein stability. This approach provides a versatile platform to functionally probe PTMs in a comparative, cell‐based context.

Phosphorylation is a prevalent post-translational modification that controls many signaling pathways by regulating protein-protein interactions. Traditionally, these interactions are studied with chemically synthesized phosphopeptides, which are often expensive and prone to dephosphorylation, or in vitro kinase reactions, which often give incomplete or off-target phosphorylation. Here, using genetic code expansion with amber-codon-directed incorporation of a nonhydrolyzable phosphoserine analog (nhpS) that is autonomously produced in E. coli, we developed a technology to produce PermaPhosPeptides and validated its utility by obtaining functional 12-mer fragments of the SARS-CoV-2 nucleocapsid protein (N) from Wuhan and later coronavirus variants. PermaPhosPeptides are phosphatase-proof and accurately mimic authentic phosphopeptides in being recognized by pS/pT-binding 14-3-3 proteins, exhibiting an average KD difference at pH 7-8 with respect to the Wuhan phosphopeptide of only 9%, as measured by fluorescence polarization. At pH 5.5, KD for the 14-3-3 complex with PermaPhosPeptide increases by 68% compared with the phosphopeptide but remains in the low micromolar range despite the charge -1 of the nhpS-group, indicating that stereochemistry of the target group is a more critical driver for 14-3-3 recognition than its precise charge. Furthermore, PermaPhosPeptides revealed consistent effects of N mutations on binding affinities for the seven human 14-3-3 isoforms, indicating specificity and sensitivity of the interaction. Given the modular genetic encoding system used, PermaPhosPeptide technology is scalable and adaptable, in principle enabling production of almost any phosphopeptide in a permanently phosphorylated form for studies by low- and high-throughput methods.

The human voltage-gated proton channel (hHv1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hHv1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands such as Zn2+. Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hHv1, we developed a Förster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hHv1 purified from E. coli. We used genetic code expansion (GCE) to generate a library of 14 full-length hHv1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hHv1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hHv1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn2+ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.

How many and which types of primary cells (domains of life) emerged from LUCA, identified employing different genetic codes.

Dendrimer-based DNA biosensor for HIV virus detection.

Understanding the genomic basis of diversification is a central goal in evolutionary biology. In recent years, the development and use of pangenomes, a genomic representation of multiple individuals within a lineage (a set of related populations, subspecies, ecotypes, or species), has enabled researchers to differentiate between DNA sequences shared by all individuals of a given lineage (core regions) from those present only in some individuals (accessory or variable regions). Differentiating between core and accessory regions has highlighted a key limitation of relying on a single reference genome: It captures the genetic code of only one individual and this biases genomic analyses and our understanding of diversification. Here, we propose that by identifying genes associated with both core and accessory regions, we can deepen our understanding of the processes underlying diversification. We suggest that analyzing pangenomes and accessory regions will provide deeper insights into diversification, hybridization, and the genetic basis of adaptation and speciation.