Genetic Code System Research Articles

7-Deaza-2'-deoxyisoguanosine, a Noncanonical Nucleoside for Nucleic Acid Code Expansion and New DNA Constructs: Nucleobase Functionalization of Inverse Watson-Crick and Purine-Purine Base Pairs.

7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids.

Over 500 natural and synthetic amino acids have been genetically encoded in the last two decades. Incorporating these noncanonical amino acids into proteins enables many powerful applications, ranging from basic research to biotechnology, materials science, and medicine. However, major challenges remain to unleash the full potential of genetic code expansion across disciplines. Here, we provide an overview of diverse genetic code expansion methodologies and systems and their final applications in prokaryotes and eukaryotes, represented by Escherichia coli and mammalian cells as the main workhorse model systems. We highlight the power of how new technologies can be first established in simple and then transferred to more complex systems. For example, whole-genome engineering provides an excellent platform in bacteria for enabling transcript-specific genetic code expansion without off-targets in the transcriptome. In contrast, the complexity of a eukaryotic cell poses challenges that require entirely new approaches, such as striving toward establishing novel base pairs or generating orthogonally translating organelles within living cells. We connect the milestones in expanding the genetic code of living cells for encoding novel chemical functionalities to the most recent scientific discoveries, from optimizing the physicochemical properties of noncanonical amino acids to the technological advancements for their in vivo incorporation. This journey offers a glimpse into the promising developments in the years to come.

A Genetic Causal Explainer for Deep Knowledge Tracing

Knowledge tracing (KT) has become an increasingly relevant problem in intelligent education services. Deep learning-based knowledge tracing (DLKT) achieves superb performance in terms of prediction accuracy, but it lacks of explainability, which makes us hard to trust or understand models. The previous work on explaining DLKT was mainly based on gradients or attention scores, which is susceptible to spurious correlations, reducing the credibility of the explanation. To address this limitation, in this paper, we propose a causal explanation method based on the genetic algorithm (GA), named Genetic Causal Explainer(GCE), which constructs a causal framework to estimate the attribution of subsequence to the predictions of DLKT models, and a genetic coding system is designed. Further, A multi-strategy initialization method inspired by domain prior knowledge is proposed, and a global empirical matrix is introduced to capture the causal correlation knowledge during the search process across instances, and guiding the mutation operators. The GCE as a post hoc explanation method can generate explanation results without affecting model training, and can be applied to analyze different DLKT models. Experimental results demonstrate the GCE perform better than other explanation methods in terms of accuracy and readability in quantitative assessments. Meanwhile, the GCE also shows good application prospects in mining educational laws and comparing KT models.

Unnatural Amino Acid Crosslinking for Increased Spatiotemporal Resolution of Chromatin Dynamics.

The utilization of an expanded genetic code and in vivo unnatural amino acid crosslinking has grown significantly in the past decade, proving to be a reliable system for the examination of protein-protein interactions. Perhaps the most utilized amino acid crosslinker, p-benzoyl-(l)-phenylalanine (pBPA), has delivered a vast compendium of structural and mechanistic data, placing it firmly in the upper echelons of protein analytical techniques. pBPA contains a benzophenone group that is activated with low energy radiation (~365 nm), initiating a diradical state that can lead to hydrogen abstraction and radical recombination in the form of a covalent bond to a neighboring protein. Importantly, the expanded genetic code system provides for site-specific encoding of the crosslinker, yielding spatial control for protein surface mapping capabilities. Paired with UV-activation, this process offers a practical means for spatiotemporal understanding of protein-protein dynamics in the living cell. The chromatin field has benefitted particularly well from this technique, providing detailed mapping and mechanistic insight for numerous chromatin-related pathways. We provide here a brief history of unnatural amino acid crosslinking in chromatin studies and outlooks into future applications of the system for increased spatiotemporal resolution in chromatin related research.

Unconventional genetic code systems in archaea.

Archaea constitute the third domain of life, distinct from bacteria and eukaryotes given their ability to tolerate extreme environments. To survive these harsh conditions, certain archaeal lineages possess unique genetic code systems to encode either selenocysteine or pyrrolysine, rare amino acids not found in all organisms. Furthermore, archaea utilize alternate tRNA-dependent pathways to biosynthesize and incorporate members of the 20 canonical amino acids. Recent discoveries of new archaeal species have revealed the co-occurrence of these genetic code systems within a single lineage. This review discusses the diverse genetic code systems of archaea, while detailing the associated biochemical elements and molecular mechanisms.

GENETIC CODING SYSTEM ANDALGEBRAIC HOLOGRAPHY

The article is devoted to the structural features of the molecular genetic coding system. These features in their matrix representation turn out to be conjugate with the matrix structures of algebraic holography, which have long been used in digital informatics. The relationship between ensembles of genetic structures and bit-reversing holography, split-quaternions, and the Poincaré disk model of hyperbolic motions is described. This connection leads to well-known works on quantum holographic noise-immune codes and makes it possible to comprehend the facts of the realization of hyperbolic geometry in genetically inherited macrophysiological phenomena.

Factors in Protobiomonomer Selection for the Origin of the Standard Genetic Code.

Natural selection of specific protobiomonomers during abiogenic development of the prototype genetic code is hindered by the diversity of structural, spatial, and rotational isomers that have identical elemental composition and molecular mass (M), but can vary significantly in their physicochemical characteristics, such as the melting temperature Tm, the Tm:M ratio, and the solubility in water, due to different positions of atoms in the molecule. These parameters differ between cis- and trans-isomers of dicarboxylic acids, spatial monosaccharide isomers, and structural isomers of α-, β-, and γ-amino acids. The stable planar heterocyclic molecules of the major nucleobases comprise four (C, H, N, O) or three (C, H, N) elements and contain a single -C=C bond and two nitrogen atoms in each heterocycle involved in C-N and C=N bonds. They exist as isomeric resonance hybrids of single and double bonds and as a mixture of tautomer forms due to the presence of -C=O and/or -NH2 side groups. They are thermostable, insoluble in water, and exhibit solid-state stability, which is of central importance for DNA molecules as carriers of genetic information. In M-Tm diagrams, proteinogenic amino acids and the corresponding codons are distributed fairly regularly relative to the distinct clusters of purine and pyrimidine bases, reflecting the correspondence between codons and amino acids that was established in different periods of genetic code development. The body of data on the evolution of the genetic code system indicates that the elemental composition and molecular structure of protobiomonomers, and their M, Tm, photostability, and aqueous solubility determined their selection in the emergence of the standard genetic code.

Recurrent sequences and the harmonic progression in genetic biomechanics

The article is devoted to the study of new approaches to the development of mathematical models in genetic biomechanics, which studies the structural relationships of the genetic coding system with genetically inherited biological forms. More specifically, we are talking about models based on the recurrent harmonic progression known in science, the universal connection of which with the information sequences of DNA heredity molecules in the genomes of higher and lower organisms was revealed in the laboratory of biomechanical systems research at the IMASH RAS. This progression is associated with fundamental hyperbolic rules for organizing DNA nucleotide sequences of genomes and is represented on a two-dimensional Cartesian plane by a hyperbolic sequence associated with natural logarithms. The latter have long been used in science to adequately model many inherited biological phenomena. The article describes previously unknown connections of natural logarithms with the structure of the molecular genetic system. The problems of modeling biological phenomena in connection with the revealed structural properties of the molecular genetic system are discussed.

Harmonic Progression in Bioinformatics and Recurrent Series in Inherited Biostructures

The article is devoted to the study of new approaches to the development of mathematical models in genetic biomechanics, which studies the structural relationships of the genetic coding system with genetically inherited biological forms. More specifically, we are talking about models based on the recurrent harmonic progression whose connection with the information sequences of DNA molecules in the genomes of higher and lower organisms was recently revealed. In particular, the article describes previously unknown connections of the function of natural logarithms with the structures of the molecular genetic system, which allow modelling the main psychophysical logarithmic law by Weber-Fechner and also many other logarithmic structures in genetically inherited biological systems. In physics, the harmonic progression is traditionally considered, first of all, as related to standing waves in resonators. Our results are correlated with Frohlich’s vibration-resonant theory about collective quantum effects and long-range communication in biological systems.

Factors of protobiomonomer selection for the emergence the genetic code

The natural selection of specific protobiomonomers during abiogenic development of the prototype genetic code is hindered by the diversity of structural, spatial, and rotational isomers. They have identical elemental composition and molecular mass (M), but can vary significantly in their physicochemical characteristics, such as melting point Tm, the Tm : M ratio, and solubility in water, due to the different arrangement of atoms in the molecule. These parameters differ between cis- and trans-isomers of dicarboxylic acids, spatial monosaccharide isomers, and structural isomers of α-, β-, and γ-amino acids. The stable planar heterocyclic molecules of the major nucleobases comprise four (C, H, N, O) or three (C, H, N) elements and contain a single -C=C bond and two nitrogen atoms in each heterocycle involved in C-N and C=N bonds. They exist as isomeric resonance hybrids of single and double bonds and as a mixture of tautomer forms due to the presence of -C=O and/or -NH2 side groups. They are thermostable, insoluble in water, and exhibit solid-state stability, which is of central importance for DNA molecules as carriers of genetic information. In M Tm diagrams, proteinogenic amino acids and the corresponding codons are distributed in a rather ordered manner relative to the distinct clusters of purine and pyrimidine bases, reflecting the correspondence between codons and amino acids that was established in different periods of genetic code development. The body of data on the evolution of the genetic coding system indicates that the elemental composition and molecular structure of protobiomonomers, and their M, Tm, photostability, and aqueous solubility determined their selection in the emergence of the standard genetic code.

Rational Design of Aptamer-Tagged tRNAs.

Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.

Nucleobase-Functionalized 5-Aza-7-deazaguanine Ribo- and 2'-Deoxyribonucleosides: Glycosylation, Pd-Assisted Cross-Coupling, and Photophysical Properties.

The special nucleobase recognition pattern of 5-aza-7-deazaguanine nucleosides makes them valuable for construction of homo purine DNA, silver-mediated base pairs, and expansion of the four letter genetic coding system. To widen the utility of 5-aza-7-deazaguanine nucleosides, side chains were introduced at position-7 of the nucleobase. As key compounds, 7-iodo nucleosides were synthesized. Nucleobase anion glycosylation of the iodo derivative of isobutyrylated 5-aza-7-deazaguanine with the bromo sugar of 2,3,5-tri-O-benzoyl-1-O-acetyl-d-ribofuranose gave the pure β-D anomeric N-9 glycosylation product (67%), whereas one-pot Vorbrüggen conditions gave only 42% of the iodinated nucleoside. The noniodinated nucleoside was formed in 84%. For the synthesis of 2'-deoxyribonucleosides, anion glycosylation performed with Hoffer's 2'-deoxyhalogenose yielded an anomeric mixture (α-D = 33% and β-D = 39%) of 2'-deoxyribonucleosides. Various side chain derivatives were prepared from nonprotected nucleosides by Pd-assisted Sonogashira or Suzuki-Miyaura cross-coupling. Among the functionalized ribonucleosides and anomeric 2'-deoxyribonucleosides, some of them showed strong fluorescence. Benzofuran and pyrene derivatives display high quantum yields in non-aqueous solvents and solvatochromism. Single-crystal X-ray analysis of 7-iodo-5-aza-7-deaza-2'-deoxyguanosine displayed intermolecular iodo-oxygen interactions in the crystal and channels filled with solvent molecules.

Phosphates as Energy Sources to Expand Metabolic Networks.

Phosphates are essential for modern metabolisms. A recent study reported a phosphate-free metabolic network and suggested that thioesters, rather than phosphates, could alleviate thermodynamic bottlenecks of network expansion. As a result, it was considered that a phosphorus-independent metabolism could exist before the phosphate-based genetic coding system. To explore the origin of phosphorus-dependent metabolism, the present study constructs a protometabolic network that contains phosphates prebiotically available using computational systems biology approaches. It is found that some primitive phosphorylated intermediates could greatly alleviate thermodynamic bottlenecks of network expansion. Moreover, the phosphorus-dependent metabolic network exhibits several ancient features. Taken together, it is concluded that phosphates played a role as important as that of thioesters during the origin and evolution of metabolism. Both phosphorus and sulfur are speculated to be critical to the origin of life.

Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli.

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

FrameOUT and FrameOUTDB: A web based application and repository for the identification and analysis of frameshift mutations

Frameshift, one of the three classes of recoding, leads to waste of energy, resources and activity of biosynthetic machinery. In addition, some peptides, probably cycotoxic synthesized after frameshifts, results in diseases and disorders like muscular dystrophies, lysosomal storage disorders, and cancer. Hidden Stop Codons that occur naturally in coding sequences among all organisms, are associated with the early termination of translation for incorrect reading frame selection and help to reduce the metabolic cost related to the frame-shift events. Hidden stop codons and their association with numerous diseases. These codons are associated with the early termination of translation for incorrect reading frame selection and help to reduce the metabolic cost related to the frame-shift events. There are lots of appearances of hidden stops in mitochondrial genomes and we tried to study this putative event in mitochondrial genomes of vertebrates. To reduce this gap, this work presents an algorithmic web based tool to study hidden stops in frame-shifted translation for vertebrate mitochondrial genomes through respective genetic code system. FrameOUT (FO), an algorithmic web based application, predicts mutations in a user input sequence, be it a diseased or a normal sequence by implementation of Hidden Markov Model. FODB is a collection of all available Frameshift events and their association with various diseases.

From quasispecies to quasispaces: coding and cooperation in chemical and electronic systems.

This contribution addresses the physical roles of spatial structures, either externally imposed or generated through self-assembly, either passive or active, on the physical chemistry of evolution. Starting with simple diffusion in closed capillaries, a one-dimensional space, it covers eight aspects of experimental and theoretical research into the interaction of evolution with spatial structures: in various dimensions, including hitherto unexplored ones, spanning from externally defined physical spaces to actively tailored spaces, assembled by the evolving components themselves. As such, it contains some original research by the author as well as tracing how other insights grew over three decades out of the mentorship of Manfred Eigen in the 1980s. Much of the early interest in spatial structures centres on its role in stabilizing higher order cooperative structures involving the coevolution of different molecules, as the genetic coding system exemplifies. Modern nanotechnology enables the design and construction of genetically encoded variants of smart components that can actively control both the proliferation of molecules and the structuring of space. A key role for this article is to show the continuity in this line of enquiry, beginning with quasispecies and projecting to autonomous microparticles with electronic genomes able to form programmable quasispaces.

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea.

ABSTRACTThe diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria (“Candidatus Parcubacteria” and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.

Precision Templated Bottom-Up Multiprotein Nanoassembly through Defined Click Chemistry Linkage to DNA.

We demonstrate an approach that allows attachment of single-stranded DNA (ssDNA) to a defined residue in a protein of interest (POI) so as to provide optimal and well-defined multicomponent assemblies. Using an expanded genetic code system, azido-phenylalanine (azF) was incorporated at defined residue positions in each POI; copper-free click chemistry was used to attach exactly one ssDNA at precisely defined residues. By choosing an appropriate residue, ssDNA conjugation had minimal impact on protein function, even when attached close to active sites. The protein-ssDNA conjugates were used to (i) assemble double-stranded DNA systems with optimal communication (energy transfer) between normally separate groups and (ii) generate multicomponent systems on DNA origami tiles, including those with enhanced enzyme activity when bound to the tile. Our approach allows any potential protein to be simply engineered to attach ssDNA or related biomolecules, creating conjugates for designed and highly precise multiprotein nanoscale assembly with tailored functionality.

Generalized crystallography, the genetic system and biochemical esthetics

The article is written for the special journal issue dedicated to Alan L. Mackay on his 90th birthday. The remarkable ideas of Alan L. Mackay about generalized crystallography, its connections with encoding of information, and also the interrelationship between art and science generate many ideas and their consequences. Mackay paid a special attention to the theme of five-fold symmetry, Fibonacci numbers and the golden section, which are essential in the structures of quasicrystals and living bodies. The present article considers a continuation of this theme in relation to the genetic coding system and to ratios of musical harmony. Hidden connections between the matrix representations the genetic code system and the golden section and the Pythagoras’s theory of esthetics of proportions are described. Such connections testify the validity of the molecular-genetic foundation of many known facts about inborn feeling of harmony in our perceptions, which becomes a part of the scientific field of “biochemical esthetics” (the term first introduced by Russian biophysicist S. Shnoll, 7). Data from theory of resonances and mathematics of noise-immunity coding of information are used in the article.

Evolution of the first genetic cells and the universal genetic code: A hypothesis based on macromolecular coevolution of RNA and proteins

A qualitative hypothesis based on coevolution of protein and nucleic acid macromolecules was developed to explain the evolution of the first genetic cells, from the likely organic chemical-rich environment of early earth, through to the Last Universal Common Ancestor (LUCA). The evolution of the first genetic cell was divided into three phases, proto-genetic cells I, II and III, and the transition to each milestone is described, based on development of chemical cross-catalysis, bio-cross-catalysis, and the universal genetic code, respectively. Selection of macromolecular properties of both peptides and nucleic acids, in response to environmental factors, was likely to be a key aspect of early evolution. The development of hereditable nucleic acids with various key functions; translation, transcription and replication, is described. These functions are envisaged to have coevolved with protein enzymes, from simple organic precursors. Genetically heritable nucleotides may have developed after the local earth environment had cooled below 63°C. Around this temperature G–C bases would have been preferentially utilized for nucleotide synthesis. Under these conditions RNA type nucleotides were then likely selected from a range of different types of nucleotide backbones through template-based synthesis. Initial development of the genetic coding system was simplified by the availability of proto-messenger RNA sequences that contained only G and C bases, and the need to encode only four amino acids. The step-wise addition of further amino acids to the code was predicted to parallel the growing metabolic complexity of the proto-genetic cell. On completion of this evolutionary process the proto-genetic cell is envisaged to have become the LUCA, the last common ancestor of bacteria, eukaryote and archaea domains. Key issues addressed by the model include: (a) the transition from non-hereditable random sequences of peptides and nucleic acids to specific proteins coded by hereditable nucleotide sequences, (b) the origin of homochiral amino acids and sugars, and (c) the mutation limits on the sizes of early nucleic acid genomes. The first genome was limited to a size of about 200 base pairs.