Structure elucidation of a multi-modular recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC 27405 and its substrate binding analysis.

Cellulases from GH9 family show endo-, exo- or processive endocellulase activity, but the reason behind the variation is unclear. A GH9 recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus was structurally characterized for conformation, binding and dynamics assessment. Modeled AtGH9C-CBM3A-CBM3B depicted (α/α)6-barrel structure with Asp98, Asp101 and Glu489 acting as catalytic triad. CD results revealed 25.2 % α-helix, 18.4 % ß-sheet and rest 56.4 % of random coils, corroborating with predictions from PSIPRED and SOPMA. MD simulation of AtGH9C-CBM3A-CBM3B bound cellotetraose showed structural stability and global compactness with lowered RMSD values (1.5 nm) as compared with only AtGH9C-CBM3A-CBM3B (1.8 nm) for 200 ns. Higher fluctuation in RMSF values in far-positioned CBM3B pointed to its redundancy in substrate binding. Docking studies showed maximum binding with cellotetraose (ΔG = -5.05 kcal/mol), with reduced affinity towards ligands with degree of polymerization (DP) lower (DP < 4) or higher than 4 (DP > 4). Processivity index displayed the enzyme to be processive with loop 3 (342-379 aa) possibly blocking the non-reducing end of cellulose chain, resulting in cellotetraose release. SAXS analysis of AtGH9C-CBM3A-CBM3B at 5 mg/mL displayed monodispersed state with fist-and-elbow shape in solution. Negative zeta potential of -24 mV at 5 mg/mL indicated stability and free from aggregation.

Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage.

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of α-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) TPT3:NaM, novel modified nucleoside triphosphates with a dual modification pattern were synthesized. We investigated the enzymatic incorporation of these nucleotide building blocks by XNA-compatible polymerases and confirmed the successful enzymatic synthesis of TPT3-modified TNA, while the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection and evolution of nuclease-resistant, high-affinity aptamers with increased chemical diversity.

Ultra-stable threose nucleic acid-based biosensors for rapid and sensitive nucleic acid detection and in vivo imaging.

The human genome's nucleotide sequence variation, such as single nucleotide mutations, can cause numerous genetic diseases. However, detecting nucleic acids accurately and rapidly in complex biological samples remains a major challenge. While natural deoxyribonucleic acid (DNA) has been used as biorecognition probes, it has limitations like poor specificity, reproducibility, nuclease-induced enzymatic degradation, and reduced bioactivity on solid surfaces. To address these issues, we introduce a stable and reliable biosensor called graphene oxide (GO)- threose nucleic acid (TNA). It comprises chemically modified TNA capture probes on GO for detecting and imaging target nucleic acids in vitro and in vivo, distinguishing single nucleobase mismatches, and monitoring dynamic changes in target microRNA (miRNA). By loading TNA capture probes onto the GO substrate, the GO-TNA sensing platform for nucleic acid detection demonstrates a significant 88-fold improvement in the detection limit compared to TNA probes alone. This platform offers a straightforward preparation method without the need for costly and labor-intensive isolation procedures or complex chemical reactions, enabling real-time analysis. The stable TNA-based GO sensing nanoplatform holds promise for disease diagnosis, enabling rapid and accurate detection and imaging of various disease-related nucleic acid molecules at the in vivo level. STATEMENT OF SIGNIFICANCE: The study's significance lies in the development of the GO-TNA biosensor, which addresses limitations in nucleic acid detection. By utilizing chemically modified nucleic acid analogues, the biosensor offers improved reliability and specificity, distinguishing single nucleobase mismatches and avoiding false signals. Additionally, its ability to detect and image target nucleic acids in vivo facilitates studying disease mechanisms. The simplified preparation process enhances practicality and accessibility, enabling real-time analysis. The biosensor's potential applications extend beyond healthcare, contributing to environmental analysis and food safety. Overall, this study's findings have substantial implications for disease diagnosis, biomedical research, and diverse applications, advancing nucleic acid detection and its impact on various fields.

Threose nucleic acid as a primitive genetic polymer and a contemporary molecular tool.

Nucleic acids serve a dual role as both genetic materials in living organisms and versatile molecular tools for various applications. Threose nuclei acid (TNA) stands out as a synthetic genetic polymer, holding potential as a primitive genetic material and as a contemporary molecular tool. In this review, we aim to provide an extensive overview of TNA research progress in these two key aspects. We begin with a retrospect of the initial discovery of TNA, followed by an in-depth look at the structural features of TNA duplex and experimental assessment of TNA as a possible RNA progenitor during early evolution of life on Earth. In the subsequent section, we delve into the recent development of TNA molecular tools such as aptamers, catalysts and antisense oligonucleotides. We emphasize the practical application of functional TNA molecules in the realms of targeted protein degradation and selective gene silencing. Our review culminates with a discussion of future research directions and the technical challenges that remain to be addressed in the field of TNA research.

A Nucleic Acid Sequence That is Catalytically Active in Both RNA and TNA Backbones.

Threose nucleic acid (TNA) is considered a potential RNA progenitor due to its chemical simplicity, base pairing property, and capability of folding into a functional tertiary structure. However, it is unknown whether the functional property can be maintained during transition from TNA to RNA. Here, we use a toggle in vitro selection to identify nucleic acid catalyst sequences that are active in both TNA and RNA backbones. One such nucleic acid enzyme with exchangeable backbone (CAMELEON) catalyzes an RNA cleavage reaction when prepared as TNA (T) and RNA (R). Further biochemical characterization reveals that CAMELEON R and T exhibit different catalytic behaviors such as rate enhancement and magnesium dependence. Structural probing and mutagenesis experiments suggest that they likely fold into distinct tertiary structures. This work demonstrates that the catalytic activity can be preserved during backbone transition from TNA to RNA and provides further experimental support for TNA as an RNA precursor in evolution.

Evolution of Functionally Enhanced α-l-Threofuranosyl Nucleic Acid Aptamers.

Synthetic genetic polymers (xeno-nucleic acids, XNAs) have the potential to transition aptamers from laboratory tools to therapeutic agents, but additional functionality is needed to compete with antibodies. Here, we describe the evolution of a biologically stable artificial genetic system composed of α-l-threofuranosyl nucleic acid (TNA) that facilitates the production of backbone- and base-modified aptamers termed "threomers" that function as high quality protein capture reagents. Threomers were discovered against two prototypical protein targets implicated in human diseases through a combination of in vitro selection and next-generation sequencing using uracil nucleotides that are uniformly equipped with aromatic side chains commonly found in the paratope of antibody-antigen crystal structures. Kinetic measurements reveal that the side chain modifications are critical for generating threomers with slow off-rate binding kinetics. These findings expand the chemical space of evolvable non-natural genetic systems to include functional groups that enhance protein target binding by mimicking the structural properties of traditional antibodies.

Synthesis and Polymerase Recognition of Threose Nucleic Acid Triphosphates Equipped with Diverse Chemical Functionalities.

Expanding the chemical space of evolvable non-natural genetic polymers (XNAs) to include functional groups that enhance protein target binding affinity offers a promising route to therapeutic aptamers with high biological stability. Here we describe the chemical synthesis and polymerase recognition of 10 chemically diverse functional groups introduced at the C-5 position of α-l-threofuranosyl uridine nucleoside triphosphate (tUTP). We show that the set of tUTP substrates is universally recognized by the laboratory-evolved polymerase Kod-RSGA. Insights into the mechanism of TNA synthesis were obtained from a high-resolution X-ray crystal structure of the postcatalytic complex bound to the primer-template duplex. A structural analysis reveals a large cavity in the enzyme active site that can accommodate the side chain of C-5-modified tUTP substrates. Our findings expand the chemical space of evolvable nucleic acid systems by providing a synthetic route to artificial genetic polymers that are uniformly modified with diversity-enhancing functional groups.

A Threose Nucleic Acid Enzyme with RNA Ligase Activity.

Threose nucleic acid (TNA) has been considered a potential RNA progenitor in evolution due to its chemical simplicity and base pairing property. Catalytic TNA sequences with RNA ligase activities might have facilitated the transition to the RNA world. Here we report the isolation of RNA ligase TNA enzymes by in vitro selection. The identified TNA enzyme T8-6 catalyzes the formation of a 2'-5' phosphoester bond between a 2',3'-diol and a 5'-triphosphate group, with a kobs of 1.1 × 10-2 min-1 (40 mM Mg2+, pH 9.0). For efficient reaction, T8-6 requires UA|GA at the ligation junction and tolerates variations at other substrate positions. Functional RNAs such as hammerhead ribozyme can be prepared by T8-6-catalyzed ligation, with site-specific introduction of a 2'-5' linkage. Together, this work provides experimental support for TNA as a plausible pre-RNA genetic polymer and also offers an alternative molecular tool for biotechnology.

Selective Prebiotic Synthesis of α-Threofuranosyl Cytidine by Photochemical Anomerization.

The structure of life's first genetic polymer is a question of intense ongoing debate. The "RNA world theory" suggests RNA was life's first nucleic acid. However, ribonucleotides are complex chemical structures, and simpler nucleic acids, such as threose nucleic acid (TNA), can carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life. The advent of any genetic polymer in life requires synthesis of its monomers. Here we demonstrate a high-yielding, stereo-, regio- and furanosyl-selective prebiotic synthesis of threo-cytidine 3, an essential component of TNA. Our synthesis uses key intermediates and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine 1. Furthermore, we demonstrate that erythro-specific 2',3'-cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine. These results suggest that TNA may have coexisted with RNA during the emergence of life.

Laboratory Observation of, Astrochemical Search for, and Structure of Elusive Erythrulose in the Interstellar Medium.

Rotational spectroscopy provides the most powerful means of identifying molecules of biological interest in the interstellar medium (ISM), but despite their importance, the detection of carbohydrates has remained rather elusive. Here, we present a comprehensive Fourier transform rotational spectroscopic study of elusive erythrulose, a sugar building block likely to be present in the ISM, employing a novel method of transferring the hygroscopic oily carbohydrate into the gas phase. The high sensitivity of the experiment allowed the rotational spectra of all monosubstituted isotopologue species of 13C-12C3H8O4 to be recorded, which, together with quantum chemical calculations, enabled us to determine their equilibrium geometries (reSE) with great precision. Searches employing the new experimental data for erythrulose have been undertaken in different ISM regions, so far including the cold areas Barnard 1, the pre-stellar core TMC-1, Sagittarius B2. Although no lines of erythrulose were found, this data will serve to enable future searches and possible detections in other ISM regions.

A multifunctional α-amylase BSGH13 from Bacillus subtilis BS-5 possessing endoglucanase and xylanase activities.

Exploring new multifunctional enzymes and understanding the mechanisms of catalytic promiscuity will be of enormous industrial and academic values. In the present study, we reported the discovery and characterization of a multifunctional enzyme BSGH13 from Bacillus subtilis BS-5. Remarkably, BSGH13 possessed α-amylase, endoglucanase, and xylanase activities. To our knowledge, this was the first report on an amylase from Bacillus species having additional endoglucanase and xylanase activities. Subsequently, we analyzed the effects of aromatic residues substitution at each site of the active site architecture on ligand-binding affinity and catalytic specificity of BSGH13 by a combination of virtual mutation and site-directed mutagenesis approaches. Our results indicated that the introduction of aromatic amino acids Phe or Trp at the positions L182 and L183 altered the local interaction network of BSGH13 towards different substrates, thus changing the multifunctional properties of BSGH13. Moreover, we provided an expanded perspective on studies of multifunctional enzymes.

Structural interpretation of the effects of threo-nucleotides on nonenzymatic template-directed polymerization.

The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3'-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands.

The development of ß-selective glycosylation reactions with benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides: enabling practical multi-gram syntheses of 4'-Thio-2'-deoxycytidine (T-dCyd) and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) to support clinical development.

The lack of effective methods to perform direct ß-selective glycosylation reactions with 2-deoxy-1,4-dithio-D-erythro-pentofuranosides has long been a significant stumbling block for the multi-gram synthesis of 4'-thio-2'-deoxy nucleosides. In addition, previously reported methods for the preparation of appropriately substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides have proven problematic for large scale synthesis. To address these issues, herein we describe the modification and optimization of previously reported methods to allow for the convenient large scale synthesis of benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides. Furthermore, we describe the development of reaction conditions for ß-selective glycosylation reactions of benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides with both N4-benzoylcytosine and 5-aza-cytosine to enable the practical multi-gram syntheses of the clinical candidates 4'-thio-2'-deoxycytidine (T-dCyd) and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd). Taken together, these new synthetic developments have made possible the preclinical and early clinical development of these important anticancer agents at the National Cancer Institute.

Synthesis and polymerase recognition of a pyrrolocytidine TNA triphosphate.

Synthetic genetics is an area of synthetic biology that aims to extend the properties of heredity and evolution to artificial genetic polymers, commonly known as xeno-nucleic acids or XNAs. In addition to establishing polymerases that are able to convert genetic information back and forth between DNA and XNA, efforts are underway to construct XNAs with expanded chemical functionality. α-L-Threose nucleic acid (TNA), a type of XNA that is recalcitrant to nuclease digestion and amenable to Darwinian evolution, provides a model system for developing XNAs with functional groups that are not present in natural DNA and RNA. Here, we describe the synthesis and polymerase activity of a cytidine TNA triphosphate analog (6-phenyl-pyrrolocytosine, tCp TP) that maintains Watson-Crick base pairing with guanine. Polymerase-mediated primer extension assays show that tCp TP is an efficient substrate for Kod-RI, a DNA-dependent TNA polymerase developed to explore the functional properties of TNA by in vitro selection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tCp TP and 7-deaza-tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available for in vitro selection.

Selection of threose nucleic acid aptamers to block PD-1/PD-L1 interaction for cancer immunotherapy.

Threose nucleic acid (TNA) aptamers were selected in vitro to bind PD-L1 protein and inhibit its interaction with PD-1. These biologically stable TNA aptamers bound target proteins with nanomolar affinities, and effectively blocked PD-1/PD-L1 interaction in vitro. After injection into a colon cancer xenograft mouse model, the TNA aptamer N5 was specifically accumulated at the tumour site, and significantly inhibited tumour growth in vivo.

Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging.

Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.

In Vitro Selection of an ATP-Binding TNA Aptamer.

Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability.

Efficient construction of a stable linear gene based on a TNA loop modified primer pair for gene delivery.

A terminal-closed linear gene with strong exonuclease resistance and serum stability was successfully constructed by polymerase chain reaction (PCR) with an α-l-threose nucleic acid (TNA) loop modified primer pair, which can be used as an efficient gene expression system in eukaryotic cells for gene delivery.

A highly constrained nucleic acid analog based on α-l-threosamine.

Chemically modified oligonucleotides (ONs) have recently gained much attention as therapeutic materials because of their improved properties. Here, a newly designed nucleic acid analog based on α-l-threosamine (named cTNA) is reported. cTNA has a "dual" constrained structure, with a bridged sugar moiety and shorter phosphoramidate backbone, to reduce the entropy loss during the hybridization. Unexpectedly, ONs containing the cTNA unit showed lower binding affinity with complementary RNA and DNA than natural ONs. Quantum chemical calculations imply that the relative nucleobase orientation of cTNA may be unfavorable for hybridization.

The mechanism of a one-substrate transketolase reaction.

Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.