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Ribonucleotides in DNA cause several types of genome instability and can be removed by ribonucleotide excision repair (RER) that is finalized by DNA ligase 1 (LIG1). However, the mechanism by which LIG1 discriminates the RER intermediate containing a 5'-RNA-DNA lesion generated by RNase H2-mediated cleavage of ribonucleotides at atomic resolution remains unknown. Here, we determine X-ray structures of LIG1/5'-rG:C at the initial step of ligation where AMP is bound to the active site of the ligase and uncover a large conformational change downstream the nick resulting in a shift at Arg(R)871 residue in the Adenylation domain of the ligase. Furthermore, we demonstrate a diminished ligation of the nick DNA substrate with a 5'-ribonucleotide in comparison to an efficient end joining of the nick substrate with a 3'-ribonucleotide by LIG1. Finally, our results demonstrate that mutations at the active site residues of the ligase and LIG1 disease-associated variants significantly impact the ligation efficiency of RNA-DNA heteroduplexes harboring "wrong" sugar at 3'- or 5'-end of nick. Collectively, our findings provide a novel atomic insight into proficient sugar discrimination by LIG1 during the processing of the most abundant form of DNA damage in cells, genomic ribonucleotides, during the initial step of the RER pathway., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (K m , 146.6 μM and V max , 0.95 μmol/min/mg) and AICAR (K m , 34.81 μM and V max , 0.56 μmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (K m , 1.81 μM and V max , 2.87 μmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 μM and 34.2 μM, respectively) compared to AICAR (Kd, 83.4 μM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The emergence of RNA on the early Earth is likely to have been influenced by chemical and physical processes that acted to filter out various alternative nucleic acids. For example, UV photostability is thought to have favored the survival of the canonical nucleotides. In a recent proposal for the prebiotic synthesis of the building blocks of RNA, ribonucleotides share a common pathway with arabino- and threo-nucleotides. We have therefore investigated non-templated primer extension with 2-aminoimidazole-activated forms of these alternative nucleotides to see if the synthesis of the first oligonucleotides might have been biased in favor of RNA. We show that non-templated primer extension occurs predominantly through 5'-5' imidazolium-bridged dinucleotides, echoing the mechanism of template-directed primer extension. Ribo- and arabino-nucleotides exhibited comparable rates and yields of non-templated primer extension, whereas threo-nucleotides showed lower reactivity. Competition experiments confirmed the bias against the incorporation of threo-nucleotides. The incorporation of an arabino-nucleotide at the end of the primer acts as a chain terminator and blocks subsequent extension. These biases, coupled with potentially selective prebiotic synthesis, and the templated copying that is known to favour the incorporation of ribonucleotides, provide a plausible model for the effective exclusion of arabino- and threo-nucleotides from primordial oligonucleotides., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities ( i.e. , dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 μm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3'-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Besides being a key player in numerous fundamental biological processes, RNA also represents a versatile platform for the creation of therapeutic agents and efficient vaccines. The production of RNA oligonucleotides, especially those decorated with chemical modifications, cannot meet the exponential demand. Due to the inherent limits of solid-phase synthesis and in vitro transcription, alternative, biocatalytic approaches are in dire need to facilitate the production of RNA oligonucleotides. Here, we present a first step towards the controlled enzymatic synthesis of RNA oligonucleotides. We have explored the possibility of a simple protection step of the vicinal cis-diol moiety to temporarily block ribonucleotides. We demonstrate that pyrimidine nucleotides protected with acetals, particularly 2',3'-O-isopropylidene, are well-tolerated by the template-independent RNA polymerase PUP (polyU polymerase) and highly efficient coupling reactions can be achieved within minutes - an important feature for the development of enzymatic de novo synthesis protocols. Even though purines are not equally well-tolerated, these findings clearly demonstrate the possibility of using cis-diol-protected ribonucleotides combined with template-independent polymerases for the stepwise construction of RNA oligonucleotides., (© 2024 Wiley-VCH GmbH.)

In an RNA world, the emergence of template-specific self-replication and catalysis necessitated the presence of motifs facilitating reliable recognition between RNA molecules. What did these motifs entail, and how did they evolve into the proteinaceous RNA recognition entities observed today? Direct observation of these primordial entities is hindered by rapid degradation over geological time scales. To overcome this challenge, researchers employ diverse approaches, including scrutiny of conserved sequences and structural motifs across extant organisms and employing directed evolution experiments to generate RNA molecules with specific catalytic abilities. In this review, we delve into the theme of ribonucleotide recognition across key periods of early Earth's evolution. We explore scenarios of RNA interacting with small molecules and examine hypotheses regarding the role of minerals and metal ions in enabling structured ribonucleotide recognition and catalysis. Additionally, we highlight instances of RNA-protein mimicry in interactions with other RNA molecules. We propose a hypothesis where RNA initially recognizes small molecules and metal ions/minerals, with subsequent mimicry by proteins leading to the emergence of proteinaceous RNA binding domains.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N 5 -CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N 5 -CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC 50 values. Several of these compounds, including the α 1 -blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

4,5-Dicyanoimidazole and 2-aminothiazole are azoles that have previously been implicated in prebiotic nucleotide synthesis. The former compound is a byproduct of adenine synthesis, and the latter compound has been shown to be capable of separating C 2 and C 3 sugars via crystallization as their aminals. We now report that the elusive intermediate cyanoacetylene can be captured by 4,5-dicyanoimidazole and accumulated as the crystalline compound N- cyanovinyl-4,5-dicyanoimidazole, thus providing a solution to the problem of concentration of atmospherically formed cyanoacetylene. Importantly, this intermediate is a competent cyanoacetylene surrogate, reacting with ribo- aminooxazoline in formamide to give ribo- anhydrocytidine ─ an intermediate in the divergent synthesis of purine and pyrimidine nucleotides. We also report a prebiotically plausible synthesis of 2-aminothiazole and examine the mechanism of its formation. The utilization of each of these azoles enhances the prebiotic synthesis of ribonucleotides, while their syntheses comport with the cyanosulfidic scenario we have previously described.

Topoisomerase 1 (Top1) incises DNA containing ribonucleotides to generate complex DNA lesions that are resolved by APE2 (Apn2 in yeast). How Apn2 engages and processes this DNA damage is unclear. Here, we report X-ray crystal structures and biochemical analysis of Apn2-DNA complexes to demonstrate how Apn2 frays and cleaves 3' DNA termini via a wedging mechanism that facilitates 1-6 nucleotide endonucleolytic cleavages. APN2 deletion and DNA-wedge mutant Saccharomyces cerevisiae strains display mutator phenotypes, cell growth defects, and sensitivity to genotoxic stress in a ribonucleotide excision repair (RER)-defective background harboring a high density of Top1-incised ribonucleotides. Our data implicate a wedge-and-cut mechanism underpinning the broad-specificity Apn2 nuclease activity that mitigates mutagenic and genome instability phenotypes caused by Top1 incision at genomic ribonucleotides incorporated by DNA polymerase epsilon., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Human PAICS is a bifunctional enzyme that is involved in the de novo purine biosynthesis, catalyzing the conversion of aminoimidazole ribonucleotide (AIR) into N -succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). It comprises two distinct active sites, AIR carboxylase (AIRc) where the AIR is initially converted to carboxyaminoimidazole ribonucleotide (CAIR) by reaction with CO 2 and SAICAR synthetase (SAICARs) in which CAIR then reacts with an aspartate to form SAICAR, in an ATP-dependent reaction. Human PAICS is a promising target for the treatment of various types of cancer, and it is therefore of high interest to develop a detailed understanding of its reaction mechanism. In the present work, density functional theory calculations are employed to investigate the PAICS reaction mechanism. Starting from the available crystal structures, two large models of the AIRc and SAICARs active sites are built and different mechanistic proposals for the carboxylation and phosphorylation-condensation mechanisms are examined. For the carboxylation reaction, it is demonstrated that it takes place in a two-step mechanism, involving a C-C bond formation followed by a deprotonation of the formed tetrahedral intermediate (known as isoCAIR) assisted by an active site histidine residue. For the phosphorylation-condensation reaction, it is shown that the phosphorylation of CAIR takes place before the condensation reaction with the aspartate. It is further demonstrated that the three active site magnesium ions are involved in binding the substrates and stabilizing the transition states and intermediates of the reaction. The calculated barriers are in good agreement with available experimental data.

AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Toward the goal of evaluation of carbocyclic ribonucleoside-containing oligonucleotide therapeutics, we developed convenient, scalable syntheses of all four carbocyclic ribonucleotide phosphoramidites and the uridine solid-support building block. Crystallographic analysis confirmed configuration and stereochemistry of these building blocks. Duplexes with carbocyclic RNA (car-RNA) modifications in one strand were less thermodynamically stable than duplexes with unmodified RNA. However, circular dichroism spectroscopy indicated that global conformations of the duplexes containing car-RNAs were similar to those in the unmodified duplexes.

In plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo., (© 2021. The Author(s).)

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Escherichia coli dnaE gene encodes the α-catalytic subunit (pol IIIα) of DNA polymerase III, the cell's main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called "steric gate" residue in the active site of the polymerase that physically clashes with the 2'-OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α-θ-ε subunits). Of the possible 38 mutants, only nine were successfully sub-cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid-encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

A mechanism of nucleoside triphosphorylation would have been critical in an evolving "RNA world" to provide high-energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. Previous reports have demonstrated that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under prebiotically-relevant conditions, but their reaction rates were unknown and the influence of reaction conditions not well-characterized. Here we established a sensitive assay that allowed for the determination of second-order rate constants for all four rNTPs, ranging from 1.7×10 -6 to 6.5×10 -6  M -1  s -1 . The ATP reaction shows a linear dependence on pH and Mg 2+ , and an enthalpy of activation of 88±4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication., (© 2021 Wiley-VCH GmbH.)

Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2'-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi-Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.

RNA structures play a fundamental role in nearly every aspect of cellular physiology and pathology. Gaining insights into the functions of RNA molecules requires accurate predictions of RNA secondary structures. However, the existing thermodynamic folding models remain less accurate than desired, even when chemical probing data, such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reactivities, are used as restraints. Unlike most SHAPE-directed algorithms that only consider SHAPE restraints for base pairing, we extract two-dimensional structural features encoded in SHAPE data and establish robust relationships between characteristic SHAPE patterns and loop motifs of various types (hairpin, internal, and bulge) and lengths (2-11 nucleotides). Such characteristic SHAPE patterns are closely related to the sugar pucker conformations of loop residues. Based on these patterns, we propose a computational method, SHAPELoop, which refines the predicted results of the existing methods, thereby further improving their prediction accuracy. In addition, SHAPELoop can provide information about local or global structural rearrangements (including pseudoknots) and help researchers to easily test their hypothesized secondary structures., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Viperin is an interferon-induced cellular protein that is conserved in animals 1 . It has previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3'-deoxy-3',4'-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase 2 . Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems.

5'-ribonucleotides including adenosine 5'-monophosphate (AMP), cytidine 5'-monophsphate (CMP), guanosine 5'-monophosphate (GMP) and uridine 5'-monophosphate (UMP) have been widely used in the food and pharmaceutical industries. This work focused on the assessment of mass transfer process and separation mechanism of four 5'-ribonucleotides and counter-ion Na + on the strong cation exchange resin NH-1. The intraparticle diffusion was determined as the rate-limiting step for the mass transfer of AMP, CMP, GMP, and Na + on the resin NH-1 through the Boyd model. Meanwhile, a homogeneous surface diffusion model (HSDM) combing ion exchange and physical adsorption was proposed and tested against adsorption kinetic data in the batch adsorption systems. The fixed-bed film-surface diffusion model based on the HSDM was then developed and successfully predicted the concentration profiles of 5'-ribonucleotides and the change of pH at the outlet of the fixed-bed in the dynamic adsorption and separation process. Finally, the separation mechanism of 5'-ribonucleotides was presented combining model prediction and experimental results. The separation of UMP, GMP and CMP were mainly based on their differences in isoelectric points, while that of AMP and CMP were lied with the discrepancy of their physical adsorption binding capacity with the resin NH-1., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest. The co-authors have reviewed the manuscript and approved it for submission to this journal for consideration., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2' modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The protocols presented in this article describe highly detailed synthesis of trifluoromethylated purine nucleotides and nucleosides (G and A). The procedure involves trifluoromethylation of properly protected (acetylated) nucleosides, followed by deprotection leading to key CF 3 -containing nucleosides. This gives synthetic access to 8-CF 3 -substituted guanosine derivatives and three adenosine derivatives (8-CF 3 , 2-CF 3 , and 2,8-diCF 3 ). In further steps, phosphorylation and phosphate elongation (for selected examples) result in respective trifluoromethylated nucleoside mono-, di-, and triphosphates. Support protocols are included for compound handling, purification procedures, analytical sample preparation, and analytical techniques used throughout the performance of the basic protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of trifluoromethylated guanosine and adenosine derivatives Basic Protocol 2: Synthesis of trifluoromethylated guanosine and adenosine monophosphates Basic Protocol 3: Synthesis of phosphorimidazolides of 8-CF3 GMP and 8-CF3 AMP Basic Protocol 4: Synthesis of trifluoromethylated guanosine and adenosine oligophosphates Support Protocol 1: TLC sample preparation and analysis Support Protocol 2: Purification protocol for Basic Protocol 1 Support Protocol 3: HPLC analysis and preparative HPLC Support Protocol 4: Ion-exchange chromatography., (© 2020 Wiley Periodicals LLC.)

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR's pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR-HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern-Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR-HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA's binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR-HSA complexes, reaffirming that AICAR binds to both site I and site II.

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.

The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and l-aspartate to N -succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Škerlová et al.)

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A previously proposed synthesis of pyrimidine ribonucleotides makes use of ultraviolet (UV) light to convert β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate, while simultaneously selectively degrading synthetic byproducts. Past studies of the photochemical reactions of pyrimidines have employed mercury arc lamps, characterized by narrowband emission centered at 254 nm, which is not representative of the UV environment of the early Earth. To further assess this process under more realistic circumstances, we investigated the wavelength dependence of the UV-driven conversion of β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate. We used constraints provided by planetary environments to assess the implications for pyrimidine nucleotides on the early Earth. We found that the wavelengths of light (255-285 nm) that most efficiently drive the deamination of β-d-ribocytidine-2',3'-cyclic phosphate to β-d-ribouridine-2',3'-cyclic phosphate are accessible on planetary surfaces such as those of the Hadean-Archaean Earth for CO 2 -N 2 -dominated atmospheres. However, continued irradiation could eventually lead to low levels of ribocytidine in a low-temperature, highly irradiated environment, if production rates are slow.

In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the last decade, it has become evident that RNA is frequently found in DNA. It is now well established that single embedded ribonucleoside monophosphates (rNMPs) are primarily introduced by DNA polymerases and that longer stretches of RNA can anneal to DNA, generating RNA:DNA hybrids. Among them, the most studied are R-loops, peculiar three-stranded nucleic acid structures formed upon the re-hybridization of a transcript to its template DNA. In addition, polyribonucleotide chains are synthesized to allow DNA replication priming, double-strand breaks repair, and may as well result from the direct incorporation of consecutive rNMPs by DNA polymerases. The bright side of RNA into DNA is that it contributes to regulating different physiological functions. The dark side, however, is that persistent RNA compromises genome integrity and genome stability. For these reasons, the characterization of all these structures has been under growing investigation. In this review, we discussed the origin of single and multiple ribonucleotides in the genome and in the DNA of organelles, focusing on situations where the aberrant processing of RNA:DNA hybrids may result in multiple rNMPs embedded in DNA. We concluded by providing an overview of the currently available strategies to study the presence of single and multiple ribonucleotides in DNA in vivo.

The oligomerization of ribonucleotides can produce short RNA strands in the absence of enzymes. This reaction gives one of two regioisomeric phosphodiester linkages, a 2',5'- or a 3',5'-diester. The former, non-natural linkage is detrimental for duplex stability, and is known to form preferentially in oligomerizations occurring in homogeneous solution with preactivated nucleotides in the presence of magnesium cations. We have studied ribonucleotide oligomerization with in situ activation, using NMR as monitoring technique. Unexpectedly, the known preference for 2',5'-linkages in the oligomerization of AMP was reversed in the absence of magnesium ions at slightly basic pH. Further, oligomerization was surprisingly efficient in the absence of Mg2+ salts, producing oligomers long enough for duplex formation. A quantitative systems chemistry analysis then revealed that the absence of magnesium ions favors the activation of nucleotides, and that the high concentration of active species can compensate for slower coupling. Further, organocatalytic intermediates can help to overcome the unfavorable regioselectivity of the magnesium-catalyzed reactions. Our findings allay concerns that RNA may have been difficult to form in the absence of enzymes. They also show that there is an efficient path to genetic material that does not require mineral surfaces or cations known to catalyze RNA hydrolysis., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2'-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors ( i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases., (© 2020 Smith et al.)

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (t D ) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold. Analyzed in mixture, the widths of the respective signals resulted in significant overlap that initially prevented their resolution on the t D scale. The separation of the four isomers was achieved by increasing the number of passes through the cIM device, which enabled to fully differentiate the characteristic ion mobility behaviors associated with very subtle structural variations. The placement of the cIM device between the mass-selective quadrupole and the time-of-flight analyzer allowed us to perform gas-phase activation of each of these ion populations, which had been first isolated according to a common mass-to-charge ratio and then separated on the basis of different ion mobility behaviors. The observed fragmentation patterns confirmed the structures of the various isomers thus substantiating the benefits of complementing unique t D information with specific fragmentation data to reach more stringent analyte identification. These capabilities were further tested by analyzing natural mono-nucleotide mixtures obtained by exonuclease digestion of total RNA extracts. In particular, the combination of cIM separation and post-mobility dissociation allowed us to establish the composition of methyl-cytidine and methyl-adenine components present in the entire transcriptome of HeLa cells. For this reason, we expect that this technique will benefit not only epitranscriptomic studies requiring the determination of identity and expression levels of RNA modifications, but also metabolomics investigations involving the analysis of natural extracts that may possibly contain subsets of isomeric/isobaric species., (© 2019 John Wiley & Sons, Ltd.)

The catalytic active site of the Polymerization Domain (PolDom) of bacterial Ligase D is designed to promote realignments of the primer and template strands and extend mispaired 3' ends. These features, together with the preferred use of ribonucleotides ( NTPs ) over deoxynucleotides (dNTPs), allow PolDom to perform efficient double strand break repair by nonhomologous end joining when only a copy of the chromosome is present and the intracellular pool of dNTPs is depleted. Here, we evaluate (i) the role of conserved histidine and serine/threonine residues in NTP insertion, and (ii) the importance in the polymerization reaction of a conserved lysine residue that interacts with the templating nucleotide. To that extent, we have analyzed the biochemical properties of variants at the corresponding His651, Ser768, and Lys606 of Pseudomonas aeruginosa PolDom ( Pa -PolDom). The results show that preferential insertion of NMPs is principally due to the histidine that also contributes to the plasticity of the active site to misinsert nucleotides. Additionally, Pa -PolDom Lys606 stabilizes primer dislocations. Finally, we show that the active site of PolDom allows the efficient use of 7,8-dihydro-8-oxo-riboguanosine triphosphate (8oxo GTP ) as substrate, a major nucleotide lesion that results from oxidative stress, inserting with the same efficiency both the anti and syn conformations of 8oxo GMP ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Currently, studies of RNA/protein interactions occupy a prominent place in molecular biology and medicine. The structures of RNA-protein complexes may be determined by X-ray crystallography or NMR for further analyses. These methods are time-consuming and difficult due to the versatility and dynamics of the RNA structure. Furthermore, due to the need to solve the "phase problem" for each dataset in crystallography, crystallographic structures of RNA are still underrepresented. Structure determination of single ribonucleotide-protein complexes is a useful tool to identify the position of single-stranded RNA-binding sites in proteins. We describe here a structural approach that incorporates affinity measurement of a protein for various single ribonucleotides, ranking the RNA/protein complexes according to their stability. This chapter describes how to perform these measurements, including a perspective for the analysis of RNA-binding sites in protein and single-nucleotide crystal soaking.

The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. DATABASE: Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively., (© 2019 Federation of European Biochemical Societies.)

Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which then can be measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation-exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases and by comparison to concentrations provided for commercial materials or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop spectrophotometry or established from droplet-digital polymerase chain reaction (ddPCR) techniques agree well with the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases or mixtures.

The presence of ribonucleotides in DNA can lead to genomic instability and cellular lethality. To prevent adventitious rNTP incorporation, the majority of the DNA polymerases (dPols) possess a steric filter. The dPol named MsDpo4 (Mycobacterium smegmatis) naturally lacks this steric filter and hence is capable of rNTP addition. The introduction of the steric filter in MsDpo4 did not result in complete abrogation of the ability of this enzyme to incorporate ribonucleotides. In comparison, DNA polymerase IV (PolIV) from Escherichia coli exhibited stringent selection for deoxyribonucleotides. A comparison of MsDpo4 and PolIV led to the discovery of an additional polar filter responsible for sugar selectivity. Thr43 represents the filter in PolIV and this residue forms interactions with the incoming nucleotide to draw it closer to the enzyme surface. As a result, the 2'-OH in rNTPs will clash with the enzyme surface, and therefore ribonucleotides cannot be accommodated in the active site in a conformation compatible with productive catalysis. The substitution of the equivalent residue in MsDpo4-Cys47, with Thr led to a drastic reduction in the ability of the mycobacterial enzyme to incorporate rNTPs. Overall, our studies evince that the polar filter serves to prevent ribonucleotide incorporation by dPols., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We described a ribonuclease-dependent cleavable beacon primer, an energy-transfer-tagged oligonucleotide inserted with a ribonucleotide, which can be cleaved by ribonuclease to generate enhanced fluorescence signals and initiate DNA amplification for single nucleotide mutation detection with ultrahigh sensitivity and selectivity.

The metabolism of imidazole-4-carboxamide (ICA) in plants has been unknown. Two metabolites ( 1 and 2 ) were isolated from ICA-treated rice, and their structures were determined by spectroscopic analysis including the single-crystal X-ray diffraction technique and synthesis. The ribotide of ICA ( 3 ), whose existence was predicted, was also synthesized and detected from the treated rice by LC-MS/MS. These results indicated that rice might interconvert ICA, 1 , and 3 to regulate the biological activity.

Terminal deoxynucleotidyl transferase (TdT), which mediates template-independent polymerization with low specificity for nucleotides, has been used for nucleotide extension of DNA oligomers. One concern is that it is difficult to control the number of incorporated nucleotides, which is a limitation on the use of TdT for single-nucleotide incorporation of DNA oligomers. Herein, we uncovered an interesting inhibitory effect on TdT when ribonucleotide substrates (rNTPs) were employed in a borate buffer. On the basis of unique inhibitory effects of the ribonucleotide-borate complex, we developed a novel enzymatic method for single-nucleotide incorporation of a DNA oligomer with a modified rNTP by TdT. Single-nucleotide incorporation of a DNA oligomer with various modified rNTPs containing an oxanine, biotin, aminoallyl or N6-propargyl group was achieved with a high yield. The 'TdT in rNTP-borate' method is quite simple and efficient for preparing a single-nucleotide modified DNA oligomer, which is useful for the design of functional DNA-based systems., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Iron (Fe) (oxyhydr)oxide minerals, which are amongst most reactive minerals in soils and sediments, are known to exhibit strong adsorption of inorganic phosphate (P i ) and organophosphate (P o ) compounds. Beyond synthetic P o compounds, much still remains unknown about the reactivity of these minerals to transform naturally-occurring P o compounds to P i , particularly with respect to solution versus surface speciation of P o hydrolysis. To investigate this reactivity with a ferrihydrite-type mineral and ribonucleotides, we employed high-resolution liquid chromatography-mass spectrometry (LC-MS), X-ray absorption near-edge structure (XANES), Fourier-transform infrared (FTIR) spectroscopy, and molecular modeling. Kinetic experiments were conducted with the mineral (1 g L -1 ) reacted with adenosine monophosphate, diphosphate, or triphosphate (respectively AMP, ADP, ATP; 50 µM). Analysis of solution organic species by LC-MS implied that only adsorption occurred with AMP and ADP but both adsorption and dephosphorylation of ATP were evident. Maximum adsorption capacities per gram of mineral were 40.6 ± 0.8 µmol AMP, 35.7 ± 1.6 µmol ADP, and 10.9 ± 1.0 µmol ATP; solution dephosphorylated by-products accounted for 15% of initial ATP. Subsequent XANES analysis of the surface species revealed that 16% of adsorbed AMP and 30% of adsorbed ATP were subjected to dephosphorylation, which was not fully quantifiable from the solution measurements. Molecular simulations predicted that ADP and ATP were complexed mainly via the phosphate groups whereas AMP binding also involved multiple hydrogen bonds with the adenosine moiety; our FTIR data confirmed these binding confirmations. Our findings thus imply that specific adsorption mechanisms dictate the recycling and subsequent trapping of P i from ribonucleotide-like biomolecules reacted with Fe (oxyhydr)oxide minerals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein-DNA interactions are important in replication, transcription, repair, as well as epigenetic modifications of DNA, which involve methylation and demethylation of DNA resulting in regulation of gene expression. Understanding of these processes and chemical tools for studying and perhaps even modulating them could be of great relevance and importance not only in chemical biology but also in real diagnostics and treatment of diseases. In the past decade, we have been working on development of synthesis of base-modified 2'-deoxyribo- or ribonucleoside triphosphates (dNTPs or NTPs) and their use in enzymatic synthesis of modified nucleic acids using DNA or RNA polymerases. These synthetic and enzymatic methods are briefly summarized with focus on recent development and outlining of scope, limitations, and further challenges. The main focus of this Account is on applications of base-modified nucleic acids in sensing of protein-DNA interactions, in covalent cross-linking to DNA-binding proteins ,and in modulation of protein-DNA binding and transcription. Several environment-sensitive fluorescent nucleotides were incorporated to DNA probes which responded to protein binding by light-up, changing of color, or lifetime of fluorescence. Using a cyclodextrin-peptide transporter, fluorescent nucleotides can be transported through the cell membrane and incorporated to genomic DNA. Several dNTPs bearing reactive groups (i.e., vinylsulfonamide or chloroacetamide) were used for polymerase synthesis of DNA reactive probes which cross-link to Cys, His, or Lys in peptides or proteins. An attractive challenge is to use DNA modifications and bioorthogonal reactions in the major groove of DNA for modulation and switching of protein-DNA interactions. We have systematically explored the influence of major-groove modifications on recognition and cleavage of DNA by restriction endonucleases and constructed simple chemical switches of DNA cleavage. Systematic study of the influence of major-groove modifications on transcription with bacterial RNA polymerases revealed not only that some modified bases are tolerated, but also that the presence of 5-hydroxymethyluracil or -cytosine can even enhance the transcription (350 or 250% compared to native DNA). Based on these results, we have constructed the first chemical switch of transcription based on photocaging of hydroxymethylpyrimidines in DNA by 2-nitrobenzyl protection (transcription off), photochemical deprotection of the DNA (transcription on), and enzymatic phosphorylation (only for 5-hydroxymethyluracil, transcription off). Although it has been so far demonstrated only in vitro, it is the proof-of-principle first step toward chemical epigenetics.

High replication fidelity, understood as the DNA polymerases’ ability to select nucleotides with both correct base and sugar, is of critical importance for maintaining the genetic stability. Due to the fact that the cellular levels of ribonucleotides are much higher than the concentrations of deoxyribonucleotides, replicative polymerases are able to incorporate ribonucleotides with up to 1000-fold higher frequency than mismatched deoxyribonucleotides. The ability to discriminate against ribonucleotides by the DNA polymerases relies on the steric gate residue in the enzyme’s catalytic centre. Despite the fact that ribonucleotides are the most abundantly inserted incorrect nucleotides in DNA, they are not observed in properly functioning cells. The major pathway responsible for the recognition and removal of ribonucleotides from DNA is called Ribonucleotide Excision Repair. The impairment of ribonucleotide removal pathways can cause increased mutation rate, replication stress, DNA breakage, problems with transcription, chromatin structure maintenance, genetic disorders and cell death. In spite of that, ribonucleotide incorporation into DNA may have some positive biological impact, stimulating mismatch repair and non-homologous end joining.

Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated. High-resolution crystal structures (<2 Å) under different illumination conditions with either FMN or 5dFMN chromophores reveal three conformations of the highly conserved glutamine 293. An allosteric hydrogen bond network linking the chromophore via Gln293 to the auxiliary A'α helix is observed. With FMN, a "flip" of the Gln293 side chain occurs between dark and lit states. 5dFMN cannot hydrogen bond through the C5 position and proved to be unable to support Au1a domain dimerization. Under blue light, the Gln293 side chain instead "swings" away in a conformation distal to the chromophore and not previously observed in existing LOV domain structures. Together, the multiple side chain conformations of Gln293 and functional analysis of 5dFMN provide new insight into the structural requirements for LOV domain activation.

Ribonucleotides can be incorporated into DNA through many different cellular processes, and abundant amounts of ribonucleotides are detected in genomic DNA. Embedded ribonucleotides lead to genomic instability through either spontaneous ribonucleotide cleavage via internal transesterification or by inducing mutagenesis, recombination, and chromosome rearrangements. Ribonucleotides misincorporated in genomic DNA can be removed by the ribonucleotide excision repair (RER) pathway in which RNase HII initiates the repair by cleaving the 5'-phosphate of the ribonucleotide. Herein, based on in vitro reconstituted nucleosome core particles (NCPs) containing a single ribonucleotide at different positions, we studied the kinetics of ribonucleotide cleavage via the internal transesterification reaction and repair of the ribonucleotides by RNase HII in NCPs. Our results show that ribonucleotide cleavage via the internal transesterification in NCPs is suppressed compared to that in free DNA. DNA bending and structural rigidity account for the suppressed ribonucleotide cleavage in NCPs. Ribonucleotide repair by RNase HII in NCPs exhibits a strong correlation between the translational and rotational positions of the ribonucleotides. An embedded ribonucleotide located at the entry site while facing outward in NCP is repaired as efficiently as that in free DNA. However, the repair of those located in the central part of NCPs and facing inward are inhibited by up to 273-fold relative to those in free dsDNA. The difference in repair efficiency appears to arise from their different accessibility to repair enzymes in NCPs. This study reveals that a ribonucleotide misincorporated in DNA assembled into NCPs is protected against cleavage. Hence, the spontaneous cleavage of the misincorporated ribonucleotides under physiological conditions is not an essential threat to the stability of chromatin DNA. Instead, their decreased repair efficiency in NCPs may result in numerous and persistent ribonucleotides in genomic DNA, which could exert other deleterious effects on DNA such as mutagenesis and recombination.

The template-directed formation of phosphodiester bonds between two nucleic acid components is a pivotal process in biology. To induce such a reaction in the absence of enzymes is a challenge. This challenge has been met for the extension of a primer with mononucleotides, but the ligation of short oligonucleotides (dimers or trimers) has proven difficult. Here we report a method for ligating dimers and trimers of ribonucleotides using in situ activation in aqueous buffer. All 16 different dimers and two trimers were tested. Binding studies by NMR showed low millimolar dissociation constants for complexes between representative dimers and hairpins mimicking primer-template duplexes, confirming that a weak template effect is not the cause of the poor ligating properties of these short oligomers. Rather, cyclization was found to compete with ligation, with up to 90% of dimer being converted to the cyclic form during the course of an assay. This side reaction is strongly sequence dependent and more pronounced for dimers than for trimers. Under optimized reaction conditions, high yields were observed with strongly pairing purines at the 3'-terminus. These results show that short oligomers of ribonucleotides are competent reactants in enzyme-free copying., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)