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.

Conformational preferences of guanine-containing threose nucleic acid building blocks in B3LYP studies.

In this paper, detailed and systematic gas-phase B3LYP conformational studies of four monomers of threose nucleic acid (TNA) with guanine attached at the C1' atom and bearing different substituents (OH, OP(=O)OH2 and OCH3) in the C2' and C3' positions of the α-l-threofuranose moiety are presented. All exocyclic single-bond (χ, ε and γ) rotations, as well as the ν0-ν4 endocyclic torsion angles, were taken into consideration. Three (threoguanosines TG1-TG3) or two (TG4) energy minima were found for the rotation about the χ torsion angle. The syn orientation (the A rotamer family) is strongly privileged in geometries TG1 and TG2, whereas the anti orientation (the C rotamer family) and the syn orientation are observed to be in equilibrium (with populations of 56% and 44%, respectively) for TG3. In the case of TG4, the high-anti orientation (the B rotamer family) turned out to be by far the most favourable, with the contribution exceeding 90% in equilibrium. Such a preference can be attributed to the inability of H-bonding between sugar and nucleobase and possibly because of the steric strains. The low-energy conformers of TG1-TG4 occupy the northeastern (P âˆ¼ 40°) and/or southern (P âˆ¼ 210°) parts of the pseudorotational wheel, which fits the A- and B-type DNA helices quite well. Additionally, in the case of TG4, some relatively stable geometries have the furanoid ring in conformation lying on the northwestern part of the pseudorotational wheel (P âˆ¼ 288°).

Enzymatic Synthesis of TNA Protects DNA Nanostructures.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3' terminus of DNA oligonucleotide, resulting in DNA-TNA chimera resistant to exonuclease digestion. Moreover, TdT-catalyzed TNA extension supports one-pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self-assembly of DNA origami nanostructures (DONs). Such TNA-protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

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.

Production of d-aldotetrose from l-erythrulose using various isomerases.

d-Aldotetroses are rare sugars that are obtained via chemical synthesis in low yield. In this study, we demonstrated that d-aldotetroses could be produced using 3 isomerases. First, l-erythrulose was epimerized using d-tagatose 3-epimerase from Pseudomonas cichorii ST-24. The specific optical rotation of the reaction solution gradually decreased to zero, indicating that approximately 50% of the l-erythrulose was converted to d-erythrulose. d, l-Erythrulose mixture was isomerized with d-arabinose isomerase from Klebsiella pneumoniae 40bXX to produce d-threose, resulting in a conversion rate of 9.35%. d-Erythrose production using l-rhamnose isomerase from Pseudomonas stutzeri LL172 resulted in a conversion rate of 12.9%. Because of the low purity of the purchased d-erythrose, the product was reduced by the Raney nickel catalyst compared with authentic erythritol. We confirmed the products using HPLC and 13C-NMR spectra. This is the first report of d-aldotetrose production using an enzymatic reaction.

Erythrose inhibits the progression to invasiveness and reverts drug resistance of cancer stem cells of glioblastoma.

Glioblastoma (GBM) is the most frequent brain cancer and more lethal than other cancers. Characteristics of this cancer are its high drug resistance, high recurrence rate and invasiveness. Invasiveness in GBM is related to overexpression of matrix metalloproteinases (MMPs) which are mediated by wnt/ß-catenin and induced by the activation of signaling pathways extracellularly activated by the cytokine neuroleukin (NLK) in cancer stem cells (CSC). Therefore, in this work we evaluated the effect of the tetrose saccharide, erythrose (Ery), a NLK inhibitor of invasiveness and drug sensitization in glioblastoma stem cells (GSC). GSC were obtained from parental U373 cell line by a CSC phenotype enrichment protocol based on microenvironmental stress conditions such as hypoxia, hipoglycemia, drug exposition and serum starvation. Enriched fraction of GSC overexpressed the typical markers of brain CSC: low CD133+ and high CD44; in addition, epithelial to mesenchyme transition (EMT) markers and MMPs were increased several times in GSC vs. U373 correlating with higher invasiveness, elongated and tubular mitochondrion and temozolomide (TMZ) resistance. IC50 of Ery was found at nM concentration and at 24 h induced a severe diminution of EMT markers, MMPs and invasiveness in GSC. Furthermore, the phosphorylation pattern of NLK after Ery exposition also was affected. In addition, when Ery was administered to GSC at subIC50, it was capable of reverting TMZ resistance at concentrations innocuous to non-tumor cancer cells. Moreover, Ery added daily induced the death of all GSC. Those findings indicated that the phytodrug Ery could be used as adjuvant therapy in GBM.

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.

An RNA-cleaving threose nucleic acid enzyme capable of single point mutation discrimination.

Threose nucleic acid has been considered a potential evolutionary progenitor of RNA because of its chemical simplicity, base pairing properties and capacity for higher-order functions such as folding and specific ligand binding. Here we report the in vitro selection of RNA-cleaving threose nucleic acid enzymes. One such enzyme, Tz1, catalyses a site-specific RNA-cleavage reaction with an observed pseudo first-order rate constant (kobs) of 0.016 min-1. The catalytic activity of Tz1 is maximal at 8 mM Mg2+ and remains relatively constant from pH 5.3 to 9.0. Tz1 preferentially cleaves a mutant epidermal growth factor receptor RNA substrate with a single point substitution, while leaving the wild-type intact. We demonstrate that Tz1 mediates selective gene silencing of the mutant epidermal growth factor receptor in eukaryotic cells. The identification of catalytic threose nucleic acids provides further experimental support for threose nucleic acid as an ancestral genetic and functional material. The demonstration of Tz1 mediating selective knockdown of intracellular RNA suggests that functional threose nucleic acids could be developed for future biomedical applications.

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.

Characterization of a GH8 ß-1,4-Glucanase from Bacillus subtilis B111 and Its Saccharification Potential for Agricultural Straws.

Herein, we cloned and expressed an endo-ß-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-ß-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50°C, respectively, and it was stable at pH 3-9 and temperature ≤50°C. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley ß-glucan, lichenin, chitosan, PASC and avicel. The Km and Vmax values of celA1805 were 1.78 mg/ml and 50.09 µmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

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.

Glycation-mediated protein crosslinking and stiffening in mouse lenses are inhibited by carboxitin in vitro.

Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.

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.