Practical Approaches to Genetic Code Expansion with Aminoacyl-tRNA Synthetase/tRNA Pairs.

Genetic code expansion (GCE) can enable the site-selective incorporation of non-canonical amino acids (ncAAs) into proteins. GCE has advanced tremendously in the last decade and can be used to create biorthogonal handles, monitor and control proteins inside cells, study post-translational modifications, and engineer new protein functions. Since establishing our laboratory, our research has focused on applications of GCE in protein and enzyme engineering using aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. This topic has been reviewed extensively, leaving little doubt that GCE is a powerful tool for engineering proteins and enzymes. Therefore, for this young faculty issue, we wanted to provide a more technical look into the methods we use and the challenges we think about in our laboratory. Since starting the laboratory, we have successfully engineered over a dozen novel aaRS/tRNA pairs tailored for various GCE applications. However, we acknowledge that the field can pose challenges even for experts. Thus, herein, we provide a review of methodologies in ncAA incorporation with some practical commentary and a focus on challenges, emerging solutions, and exciting developments.

Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.

Inhibition and structural insights of leishmanial glutamyl-tRNA synthetase for designing potent therapeutics.

Aminoacyl-tRNA synthetases (aaRSs), essential components of the protein synthesizing machinery, have been often chosen for devising therapeutics against parasitic diseases. Due to their relevance in drug development, the current study was designed to explore functional and structural aspects of Leishmania donovani glutamyl-tRNA synthetase (LdGluRS). Hence, LdGluRS was cloned into an expression vector and purified to homogeneity using chromatographic techniques. Purified protein showed maximum enzymatic activity at physiological pH, with more binding capacity towards its cofactor (Adenosine triphosphate, 0.06 ± 0.01 mM) than the cognate substrate (L-glutamate, 9.5 ± 0.5 mM). Remarkably, salicylate inhibited LdGluRS competitively with respect to L-glutamate and exhibited druglikeness with negligible effect on human macrophages. The protein possessed more α-helices (43 %) than ß-sheets (12 %), whereas reductions in thermal stability and cofactor-binding affinity, along with variation in mode of inhibition after mutation signified the role of histidine (H60) as a catalytic residue. LdGluRS could also generate a pro-inflammatory milieu in human macrophages by upregulating cytokines. The docking study demonstrated the placement of salicylate into LdGluRS substrate-binding site, and the complex was found to be stable during molecular dynamics (MD) simulation. Altogether, our study highlights the understanding of molecular inhibition and structural features of glutamyl-tRNA synthetase from kinetoplastid parasites.

Enzymic recognition of amino acids drove the evolution of primordial genetic codes.

How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.

Enzyme redesign and genetic code expansion.

Enzyme design is an important application of computational protein design (CPD). It can benefit enormously from the additional chemistries provided by noncanonical amino acids (ncAAs). These can be incorporated into an 'expanded' genetic code, and introduced in vivo into target proteins. The key step for genetic code expansion is to engineer an aminoacyl-transfer RNA (tRNA) synthetase (aaRS) and an associated tRNA that handles the ncAA. Experimental directed evolution has been successfully used to engineer aaRSs and incorporate over 200 ncAAs into expanded codes. But directed evolution has severe limits, and is not yet applicable to noncanonical AA backbones. CPD can help address several of its limitations, and has begun to be applied to this problem. We review efforts to redesign aaRSs, studies that designed new proteins and functionalities with the help of ncAAs, and some of the method developments that have been used, such as adaptive landscape flattening Monte Carlo, which allows an enzyme to be redesigned with substrate or transition state binding as the design target.

Mammalian trans-editing factor ProX is able to deacylate tRNAThr mischarged with alanine.

The precise coupling of tRNAs with their cognate amino acids, known as tRNA aminoacylation, is a stringently regulated process that governs translation fidelity. To ensure fidelity, organisms deploy multiple layers of editing mechanisms to correct mischarged tRNAs. Prior investigations have unveiled the propensity of eukaryotic AlaRS to erroneously attach alanine onto tRNACys and tRNAThr featuring the G4:U69 base pair. In light of this, and given ProXp-ala's capacity in deacylating Ala-tRNAPro, we embarked on exploring whether this trans-editing factor could extend its corrective function to encompass these mischarged tRNAs. Our in vitro deacylation assays demonstrate that murine ProXp-ala (mProXp-ala) is able to efficiently hydrolyze Ala-tRNAThr, while Ala-tRNACys remains unaffected. Subsequently, we determined the first structure of eukaryotic ProXp-ala, revealing a dynamic helix α2 involved in substrate binding. By integrating molecular dynamics simulations and biochemical assays, we pinpointed the pivotal interactions between mProXp-ala and Ala-tRNA, wherein the basic regions of mProXp-ala as well as the C3-G70 plays essential role in recognition. These observations collectively provide a cogent rationale for mProXp-ala's deacylation proficiency against Ala-tRNAThr. Our findings offer valuable insights into the translation quality control within higher eukaryotic organisms, where the fidelity of translation is safeguarded by the multi-functionality of extensively documented proteins.

Optimization of genetic code expansion in the baculovirus expression vector system (BEVS).

The production of recombinant proteins containing unnatural amino acids, commonly known as genetic code expansion (GCE), represents a breakthrough in protein engineering that allows for the creation of proteins having novel designed properties. The naturally occurring orthogonal pyrrolysine tRNA/aminoacyl-tRNApyl synthetase pair (tRNApyl/PylRS) found in Methanosarcinaceae species has provided a rich platform for protein engineers to build a library of amino acid derivatives suitable for the introduction of novel chemical functionalities. While reports of the production of such recombinant proteins utilizing the tRNApyl/PylRS pair, or mutants thereof, is commonplace in Escherichia coli and mammalian cell expression systems, there has only been a single such report of GCE in the other stalwart of recombinant protein production, the baculovirus expression vector system (BEVS). However, that report formulates protein production within the designs of the MultiBac expression system [1]. The current study frames protein production within the strategies of the more commonplace Bac-to-Bac system of recombinant baculovirus production, via the development of novel baculovirus transfer vectors that harbor the tRNApyl/PylRS pair. The production of recombinant proteins harboring an unnatural amino acid(s) was examined using both an in cis and an in trans arrangement of the tRNApyl/PylRS pair relative to the target protein ORF i.e. the latter resides, respectively, on either the same vector as the tRNApyl/PylRS pair, or on a separate vector and deployed in a viral co-infection experiment. Aspects of the transfer vector designs and the viral infection conditions were investigated.

Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs.

Mutually orthogonal aminoacyl transfer RNA synthetase/transfer RNA pairs provide a foundation for encoding non-canonical amino acids into proteins, and encoded non-canonical polymer and macrocycle synthesis. Here we discover quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/pyrrolysyl-tRNA (tRNAPyl) pairs. We discover empirical sequence identity thresholds for mutual orthogonality and use these for agglomerative clustering of PylRS and tRNAPyl sequences; this defines numerous sequence clusters, spanning five classes of PylRS/tRNAPyl pairs (the existing classes +N, A and B, and newly defined classes C and S). Most of the PylRS clusters belong to classes that were unexplored for orthogonal pair generation. By testing pairs from distinct clusters and classes, and pyrrolysyl-tRNAs with unusual structures, we resolve 80% of the pairwise specificities required to make quintuply orthogonal PylRS/tRNAPyl pairs; we control the remaining specificities by engineering and directed evolution. Overall, we create 924 mutually orthogonal PylRS/tRNAPyl pairs, 1,324 triply orthogonal pairs, 128 quadruply orthogonal pairs and 8 quintuply orthogonal pairs. These advances may provide a key foundation for encoded polymer synthesis.

Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl-tRNA synthetase 1.

Prolyl-tRNA synthetase 1 (PARS1) has attracted much interest in controlling pathologic accumulation of collagen containing high amounts of proline in fibrotic diseases. However, there are concerns about its catalytic inhibition for potential adverse effects on global protein synthesis. We developed a novel compound, DWN12088, whose safety was validated by clinical phase 1 studies, and therapeutic efficacy was shown in idiopathic pulmonary fibrosis model. Structural and kinetic analyses revealed that DWN12088 binds to catalytic site of each protomer of PARS1 dimer in an asymmetric mode with different affinity, resulting in decreased responsiveness at higher doses, thereby expanding safety window. The mutations disrupting PARS1 homodimerization restored the sensitivity to DWN12088, validating negative communication between PARS1 promoters for the DWN12088 binding. Thus, this work suggests that DWN12088, an asymmetric catalytic inhibitor of PARS1 as a novel therapeutic agent against fibrosis with enhanced safety.

Targeting Aminoacyl tRNA Synthetases for Antimalarial Drug Development.

Infections caused by malaria parasites place an enormous burden on the world's poorest communities. Breakthrough drugs with novel mechanisms of action are urgently needed. As an organism that undergoes rapid growth and division, the malaria parasite Plasmodium falciparum is highly reliant on protein synthesis, which in turn requires aminoacyl-tRNA synthetases (aaRSs) to charge tRNAs with their corresponding amino acid. Protein translation is required at all stages of the parasite life cycle; thus, aaRS inhibitors have the potential for whole-of-life-cycle antimalarial activity. This review focuses on efforts to identify potent plasmodium-specific aaRS inhibitors using phenotypic screening, target validation, and structure-guided drug design. Recent work reveals that aaRSs are susceptible targets for a class of AMP-mimicking nucleoside sulfamates that target the enzymes via a novel reaction hijacking mechanism. This finding opens up the possibility of generating bespoke inhibitors of different aaRSs, providing new drug leads.

Study of the Allosteric Mechanism of Human Mitochondrial Phenylalanyl-tRNA Synthetase by Transfer Entropy via an Improved Gaussian Network Model and Co-evolution Analyses.

We propose an improved transfer entropy approach called the dynamic version of the force constant fitted Gaussian network model based on molecular dynamics ensemble (dfcfGNMMD) to explore the allosteric mechanism of human mitochondrial phenylalanyl-tRNA synthetase (hmPheRS), one of the aminoacyl-tRNA synthetases that play a crucial role in translation of the genetic code. The dfcfGNMMD method can provide reliable estimates of the transfer entropy and give new insights into the role of the anticodon binding domain in driving the catalytic domain in aminoacylation activity and into the effects of tRNA binding and residue mutation on the enzyme activity, revealing the causal mechanism of the allosteric communication in hmPheRS. In addition, we incorporate the residue dynamic and co-evolutionary information to further investigate the key residues in hmPheRS allostery. This study sheds light on the mechanisms of hmPheRS allostery and can provide important information for related drug design.

Engineering mutually orthogonal PylRS/tRNA pairs for dual encoding of functional histidine analogues.

The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here, we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRSIFGFF ) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF ) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and x-ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3-(3-pyridyl)-L-alanine (3-Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3-Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.

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.

Computational Methods for Molecular Understanding of the Antibiotic-Aminoacyl tRNA Synthetase Interaction.

Developing an understanding of the interactions between an antibiotic and its binding site in a pathogen cell is the key to antibiotic design-an important cost-saving methodology compared to the costly and time-consuming random trial-and-error approach. The rapid development of antibiotic resistance provides an impetus for such studies. Recent years have witnessed the beginning of the use of combined computational techniques, including computer simulations and quantum mechanical computations, to understand how antibiotics bind at the active site of aminoacyl tRNA synthetases (aaRSs) from pathogens. Such computational protocols assist the knowledge-based design of antibiotics targeting aaRSs, which are their validated targets. After the ideas behind the protocols and their strategic planning are discussed, the protocols are described along with their major outcomes. This is followed by an integration of results from the different basic protocols. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Analysis of active-site residues from primary sequence of synthetase and transfer RNAs Basic Protocol 2: Molecular dynamics simulation-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex Basic Protocol 3: Quantum mechanical method-based protocol to study the structure and dynamics of the aaRS active site:antibiotic complex.

The binding mode of orphan glycyl-tRNA synthetase with tRNA supports the synthetase classification and reveals large domain movements.

As a class of essential enzymes in protein translation, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) are organized into two classes of 10 enzymes each, based on two conserved active site architectures. The (αß)2 glycyl-tRNA synthetase (GlyRS) in many bacteria is an orphan aaRS whose sequence and unprecedented X-shaped structure are distinct from those of all other aaRSs, including many other bacterial and all eukaryotic GlyRSs. Here, we report a cocrystal structure to elucidate how the orphan GlyRS kingdom specifically recognizes its substrate tRNA. This structure is sharply different from those of other aaRS-tRNA complexes but conforms to the clash-free, cross-class aaRS-tRNA docking found with conventional structures and reinforces the class-reconstruction paradigm. In addition, noteworthy, the X shape of orphan GlyRS is condensed with the largest known spatial rearrangement needed by aaRSs to capture tRNAs, which suggests potential nonactive site targets for aaRS-directed antibiotics, instead of less differentiated hard-to-drug active site locations.

Update of the Pyrrolysyl-tRNA Synthetase/tRNAPyl Pair and Derivatives for Genetic Code Expansion.

The cotranslational incorporation of pyrrolysine (Pyl), the 22nd proteinogenic amino acid, into proteins in response to the UAG stop codon represents an outstanding example of natural genetic code expansion. Genetic encoding of Pyl is conducted by the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNAPyl. Owing to the high tolerance of PylRS toward diverse amino acid substrates and great orthogonality in various model organisms, the PylRS/tRNAPyl-derived pairs are ideal for genetic code expansion to insert noncanonical amino acids (ncAAs) into proteins of interest. Since the discovery of cellular components involved in the biosynthesis and genetic encoding of Pyl, synthetic biologists have been enthusiastic about engineering PylRS/tRNAPyl-derived pairs to rewrite the genetic code of living cells. Recently, considerable progress has been made in understanding the molecular phylogeny, biochemical properties, and structural features of the PylRS/tRNAPyl pair, guiding its further engineering and optimization. In this review, we cover the basic and updated knowledge of the PylRS/tRNAPyl pair's unique characteristics that make it an outstanding tool for reprogramming the genetic code. In addition, we summarize the recent efforts to create efficient and (mutually) orthogonal PylRS/tRNAPyl-derived pairs for incorporation of diverse ncAAs by genome mining, rational design, and advanced directed evolution methods.

Solution X-ray scattering highlights discrepancies in Plasmodium multi-aminoacyl-tRNA synthetase complexes.

tRip is a tRNA import protein specific to Plasmodium, the causative agent of malaria. In addition to its membrane localization and tRNA trafficking properties, tRip has the capacity to associate with three aminoacyl-tRNA synthetases (aaRS), the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases. In eukaryotes, such multi-aaRSs complexes (MSC) regulate the moonlighting activities of aaRSs. In Plasmodium, tRip and the three aaRSs all contain an N-terminal GST-like domain involved in the assembly of two independent complexes: the Q-complex (tRip:ERS:QRS) and the M-complex (tRip:ERS:MRS) with a 2:2:2 stoichiometry and in which the association of the GST-like domains of tRip and ERS (tRip-N:ERS-N) is central. In this study, the crystal structure of the N-terminal GST-like domain of ERS was solved and made possible further investigation of the solution architecture of the Q- and M-complexes by small-angle x-ray scattering (SAXS). This strategy relied on the engineering of a tRip-N-ERS-N chimeric protein to study the structural scaffold of both Plasmodium MSCs and confirm the unique homodimerization pattern of tRip in solution. The biological impact of these structural arrangements is discussed.

Tyrosine-targeted covalent inhibition of a tRNA synthetase aided by zinc ion.

Aminoacyl-tRNA synthetases (AARSs), a family of essential protein synthesis enzymes, are attractive targets for drug development. Although several different types of AARS inhibitors have been identified, AARS covalent inhibitors have not been reported. Here we present five unusual crystal structures showing that threonyl-tRNA synthetase (ThrRS) is covalently inhibited by a natural product, obafluorin (OB). The residue forming a covalent bond with OB is a tyrosine in ThrRS active center, which is not commonly modified by covalent inhibitors. The two hydroxyl groups on the o-diphenol moiety of OB form two coordination bonds with the conserved zinc ion in the active center of ThrRS. Therefore, the ß-lactone structure of OB can undergo ester exchange reaction with the phenolic group of the adjacent tyrosine to form a covalent bond between the compound and the enzyme, and allow its nitrobenzene structure to occupy the binding site of tRNA. In addition, when this tyrosine was replaced by a lysine or even a weakly nucleophilic arginine, similar bonds could also be formed. Our report of the mechanism of a class of AARS covalent inhibitor targeting multiple amino acid residues could facilitate approaches to drug discovery for cancer and infectious diseases.

Fundamentals behind the specificity of Cysteinyl-tRNA synthetase: MD and QM/MM joint investigations.

Cysteinyl-tRNA synthetase (CysRS) catalyzes the aminoacylation reaction of cysteine to its cognate tRNACys in the first step of protein translation. It is found that CysRS is different from other aaRSs as it transfers cysteine without the need for an editing reaction, which is not applicable in the case of serine despite the similarity in their structures. Surprisingly, the reasons why CysRS has high amino acid specificity are not clear yet. In this research, the binding configurations of Cys-AMP and its near-cognate amino acid Ser-AMP with CysRS are compared by Molecular Dynamics (MD). The results reveal that CysRS screens the substrate Cys-AMP to a certain extent in the process of combination and recognition, thus providing a guarantee for the high selectivity of the next reaction. While Ser-AMP is in a folded state in CysRS. In the meanwhile, the interaction between Cys-AMP and Zn963 in CysRS is much stronger than Ser-AMP. The substrate-assisted aminoacylation mechanism in CysRS is also explored by Quantum Mechanics/Molecular Mechanics (QM/MM) modeling. According to the QM/MM potential energies, the energy barrier of TSCys-AMP is 91.75 kJ/mol, while that of TSSer-AMP is close to 150 kJ/mol. Based on thermochemistry calculations, it is found that the product of Cys-AMP is more stable than the reactant. In contrast, Ser-AMP has a reactant that is more stable than its product. As a result, it reflects that the specificity of CysRS originates from both the kinetic and thermodynamical perspectives of the reaction. Our investigations demonstrate comprehensively on how CysRS recognizes and catalyzes the substrate Cys-AMP, hoping to provide some guidance for researchers in this area.

Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles.

The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains. We define 49 engineered mutually orthogonal pairs that recognize distinct non-canonical amino acids or alpha hydroxy acids and decode distinct codons. Finally, we combine our advances to programme Syn61Δ3-derived cells for the encoded synthesis of 12 diverse non-natural depsipeptide macrocycles, which contain two non-canonical side chains and either one or two ester bonds.