Nucleotide addition and cleavage by RNA polymerase II: Coordination of two catalytic reactions using a single active site.

RNA polymerase II (Pol II) incorporates complementary ribonucleotides into the growing RNA chain one at a time via the nucleotide addition cycle. The nucleotide addition cycle, however, is prone to misincorporation of noncomplementary nucleotides. Thus, to ensure transcriptional fidelity, Pol II backtracks and then cleaves the misincorporated nucleotides. These two reverse reactions, nucleotide addition and cleavage, are catalyzed in the same active site of Pol II, which is different from DNA polymerases or other endonucleases. Recently, substantial progress has been made to understand how Pol II effectively performs its dual role in the same active site. Our review highlights these recent studies and provides an overall model of the catalytic mechanisms of Pol II. In particular, RNA extension follows the two-metal-ion mechanism, and several Pol II residues play important roles to facilitate the catalysis. In sharp contrast, the cleavage reaction is independent of any Pol II residues. Interestingly, Pol II relies on its residues to recognize the misincorporated nucleotides during the backtracking process, prior to cleavage. In this way, Pol II efficiently compartmentalizes its two distinct catalytic functions using the same active site. Lastly, we also discuss a new perspective on the potential third Mg2+ in the nucleotide addition and intrinsic cleavage reactions.

Nucleotide analogues containing a pyrrolidine, piperidine or piperazine ring: Synthesis and evaluation of inhibition of plasmodial and human 6-oxopurine phosphoribosyltransferases and in vitro antimalarial activity.

Parasites of the Plasmodium genus are unable to produce purine nucleotides de novo and depend completely on the salvage pathway. This fact makes plasmodial hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT] a valuable target for development of antimalarial agents. A series of nucleotide analogues was designed, synthesized and evaluated as potential inhibitors of Plasmodium falciparum HGXPRT, P. vivax HGPRT and human HGPRT. These novel nucleoside phosphonates have a pyrrolidine, piperidine or piperazine ring incorporated into the linker connecting the purine base to a phosphonate group(s) and exhibited a broad range of Ki values between 0.15 and 72 µM. The corresponding phosphoramidate prodrugs, able to cross cell membranes, have been synthesized and evaluated in a P. falciparum infected human erythrocyte assay. Of the eight prodrugs evaluated seven exhibited in vitro antimalarial activity with IC50 values within the range of 2.5-12.1 µM. The bis-phosphoramidate prodrug 13a with a mean (SD) IC50 of 2.5 ± 0.7 µM against the chloroquine-resistant P. falciparum W2 strain exhibited low cytotoxicity in the human hepatocellular liver carcinoma (HepG2) and normal human dermal fibroblasts (NHDF) cell lines at a concentration of 100 µM suggesting good selectivity for further structure-activity relationship investigations.

Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly.

The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III2+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH) potently increased respirasome assembly and activity. By-passing DHODH inhibition via uridine supplementation decreases SC assembly by altering mitochondrial phospholipid composition, specifically elevated peroxisomal-derived ether phospholipids. Cell growth rates upon DHODH inhibition depend on ether lipid synthesis and SC assembly. These data reveal that nucleotide pools signal to peroxisomes to modulate synthesis and transport of ether phospholipids to mitochondria for SC assembly, which are necessary for optimal cell growth in conditions of nucleotide limitation.

4-Substituted-1,2,3-triazolo nucleotide analogues as CD73 inhibitors, their synthesis, in vitro screening, kinetic and in silico studies.

Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-ß-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P-C-P-C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The ß-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds (2a and 2b) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.

Highly convenient and highly specific-and-sensitive PCR using Se-atom modified dNTPs.

Primer design and condition optimization for PCR are tedious and labour-intensive. To conveniently achieve high selectivity, sensitivity and robustness, herein, we first report a new strategy with Se-dNTPs to enhance PCR specificity (over 240-fold) and sensitivity (up to single-digit), effectively eliminating non-specific products and simplifing PCR design and optimization.

COSMO-RS-Based Descriptors for the Machine Learning-Enabled Screening of Nucleotide Analogue Drugs against SARS-CoV-2.

Chemical similarity-based approaches employed to repurpose or develop new treatments for emerging diseases, such as COVID-19, correlates molecular structure-based descriptors of drugs with those of a physiological counterpart or clinical phenotype. We propose novel descriptors based on a COSMO-RS (short for conductor-like screening model for real solvents) σ-profiles for enhanced drug screening enabled by machine learning (ML). The descriptors' performance is hereby illustrated for nucleotide analogue drugs that inhibit the ribonucleic acid-dependent ribonucleic acid polymerase, key to viral transcription and genome replication. The COSMO-RS-based descriptors account for both chemical reactivity and structure, and are more effective for ML-based screening than fingerprints based on molecular structure and simple physical/chemical properties. The descriptors are evaluated using principal component analysis, an unsupervised ML technique. Our results correlate with the active monophosphate forms of the leading drug remdesivir and the prospective drug EIDD-2801 with nucleotides, followed by other promising drugs, and are superior to those from molecular structure-based descriptors and molecular docking. The COSMO-RS-based descriptors could help accelerate drug discovery for the treatment of emerging diseases.

Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase.

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

Synthesis and Biochemical Evaluation of Noncyclic Nucleotide Exchange Proteins Directly Activated by cAMP 1 (EPAC1) Regulators.

Exchange proteins directly activated by cAMP (EPAC) play a central role in various biological functions, and activation of the EPAC1 protein has shown potential benefits for the treatment of various human diseases. Herein, we report the synthesis and biochemical evaluation of a series of noncyclic nucleotide EPAC1 activators. Several potent EPAC1 binders were identified including 25g, 25q, 25n, 25u, 25e, and 25f, which promote EPAC1 guanine nucleotide exchange factor activity in vitro. These agonists can also activate EPAC1 protein in cells, where they exhibit excellent selectivity toward EPAC over protein kinase A and G protein-coupled receptors. Moreover, 25e, 25f, 25n, and 25u exhibited improved selectivity toward activation of EPAC1 over EPAC2 in cells. Of these, 25u was found to robustly inhibit IL-6-activated signal transducer and activator of transcription 3 (STAT3) and subsequent induction of the pro-inflammatory vascular cell adhesion molecule 1 (VCAM1) cell-adhesion protein. These novel EPAC1 activators may therefore act as useful pharmacological tools for elucidation of EPAC function and promising drug leads for the treatment of relevant human diseases.

Codon Directional Asymmetry Suggests Swapped Prebiotic 1st and 2nd Codon Positions.

Background: Codon directional asymmetry (CDA) classifies the 64 codons into palindromes (XYX, CDA = 0), and 5'- and 3'-dominant (YXX and XXY, CDA < 0 and CDA > 0, respectively). Previously, CDA was defined by the purine/pyrimidine divide (A,G/C,T), where X is either a purine or a pyrimidine. For the remaining codons with undefined CDA, CDA was defined by the 5' or 3' nucleotide complementary to Y. This CDA correlates with cognate amino acid tRNA synthetase classes, antiparallel beta sheet conformation index and the evolutionary order defined by the self-referential genetic code evolution model (CDA < 0: class I, high beta sheet index, late genetic code inclusion). Methods: We explore associations of CDAs defined by nucleotide classifications according to complementarity strengths (A:T, weak; C:G, strong) and keto-enol/amino-imino groupings (G,T/A,C), also after swapping 1st and 2nd codon positions with amino acid physicochemical and structural properties. Results: Here, analyses show that for the eight codons whose purine/pyrimidine-based CDA requires using the rule of complementarity with the midposition, using weak interactions to define CDA instead of complementarity increases associations with tRNA synthetase classes, antiparallel beta sheet index and genetic code evolutionary order. CDA defined by keto-enol/amino-imino groups, 1st and 2nd codon positions swapped, correlates with amino acid parallel beta sheet formation indices and Doolittle's hydropathicities. Conclusions: Results suggest (a) prebiotic swaps from N2N1N3 to N1N2N3 codon structures, (b) that tRNA-mediated translation replaced direct codon-amino acid interactions, and (c) links between codon structures and cognate amino acid properties.

Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.

Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

Extensive free-energy simulations identify water as the base in nucleotide addition by DNA polymerase.

Transphosphorylation of nucleotide triphosphates is the central reaction in DNA replication by DNA polymerase as well as many other biological processes. Despite its importance, the microscopic chemical mechanism of transphosphorylation of nucleotide triphosphates is, in most cases, unknown. Here we use extensive simulations of DNA polymerase η to test mechanistic hypotheses. We systematically survey the reactive space by calculating 2D free-energy surfaces for 10 different plausible mechanisms that have been proposed. We supplement these free-energy surfaces with calculations of pKa for a number of potentially acidic protons in different states relevant to the catalytic cycle. We find that among all of the conditions that we test, the smallest activation barrier occurs for a reaction where a Mg2+-coordinated water deprotonates the nucleophilic 3'-OH, and this deprotonation is concerted with the phosphoryl transfer. The presence of a third Mg2+ in the active site lowers the activation barrier for the water-as-base mechanism, as does protonation of the pyrophosphate leaving group, which is consistent with general acid catalysis. The results demonstrate the value of simulations, when used in conjunction with experimental data, to help establish a microscopic chemical mechanism in a complex environment.

Label-free discrimination of single nucleotide changes in DNA by reflectometric interference Fourier transform spectroscopy.

Phenotypic variation - such as disease susceptibility and differential drug response - has a strong genetic component. Substantial effort has therefore been made to identify causal genomic variants explaining such variation among humans. Point mutations (PMs), which are single nucleotide changes in the genome, have been identified to be the most abundant form of causal genomic variants, making them useful, reliable diagnostic markers. Methods developed to genotype PMs have moved towards solid-phase assays, which not only show greater sensitivity and specificity, but also enable scalability and faster processing time. Most current assays are, however, based on fluorescent probes, which makes them relatively expensive. To develop a more cost-effective label-free genotyping method, we used a porous silicon (PSi) base as an efficient support for DNA biosensing and coupled it with reflectometric interference Fourier transform spectroscopy (RIFTS). To assess the versatility of this approach, we tested both a single nucleotide substitution in VKORC1 (-1639G > A; rs9923231) and a single nucleotide insertion in BRCA1 (5382insC; rs80357906). We demonstrate that the PSi-RIFTS method can efficiently detect both PM types with high sensitivity where hybridization of complementary DNA can be quantifiably differentiated from mismatch and non-complementary hybridization events. In addition, we show that the PSi base with immobilized DNA not only can be re-used to type further samples, but it also remains stable for 14 days, suggesting its potential for high-throughput applications.

Synthesis of ABBV-168, a 2'-Bromouridine for the Treatment of Hepatitis C.

ABBV-168 is a dihalogenated nucleotide under investigation for the treatment of hepatitis C virus. Three synthetic routes aimed at achieving the stereoselective installation of the C2' gem-Br,F substitution and subsequent Vorbruggen glycosylation were explored to prepare the penultimate nucleoside intermediate. Development culminated in a route to ABBV-168 featuring a de novo chromatography-free furanose synthesis, protecting group-directed Vorbruggen glycosylation, and highly selective phosphoramidation to furnish the API.

OsCML16 interacts with a novel CC-NBS-LRR protein OsPi304 in the Ca2+/Mg2+ dependent and independent manner in rice.

In plants, many target proteins of calmodulins (CaMs) have been identified in cellular metabolism and responses. However, calmodulin-like proteins (CMLs) and their target proteins have not been discovered in stress responses in rice. In this study, a novel CC-NBS-LRR protein was obtained in screening a cold stress rice seedlings yeast cDNA library with OsCML16 as bait. Furthermore, yeast two-hybrid and BiFC assays demonstrated that the full length, CC region in the N-terminus and LRR in the C-terminus of Pi304 protein could interact with OsCML16. More interestingly, OsCML16 bound to the 1-10 motif rather than 1-14 motif in the Ca2+ or Mg2+ dependent manner in vitro. In addition, transcript levels of OsCML16 and OsPi304 were induced more markedly in Nipponbare than in 9311 under cold stress. Taken together, these data indicates that they are involved in the cold stress signaling and response in rice.

Direct Determination of Six Cytokinin Nucleotide Monophosphates in Coconut Flesh by Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry.

Coconut contains many uncharacterized cytokinins that have important physiological effects in plants and humans. In this work, a method based on liquid chromatography-tandem mass spectrometry was developed for identification and quantification of six cytokinin nucleotide monophosphates in coconut flesh. Excellent separation was achieved using a low-coverage C18 bonded-phase column with an acidic mobile phase, which greatly improved the retention of target compounds. To enable high-throughput analysis, a single-step solid-phase extraction using mixed-mode anion-exchange cartridges was employed for sample preparation. This proved to be an effective method to minimize matrix effects and ensure high selectivity. The limits of detection varied from 0.06 to 0.3 ng/mL, and the limits of quantification ranged from 0.2 to 1.0 ng/mL. The linearity was statistically verified over 2 orders of magnitude, giving a coefficient of determination (R2) greater than 0.9981. The mean recoveries were from 81 to 108%; the intraday precision (n = 6) was less than 11%; and the interday precision (n = 11) was within 14%. The developed method was applied to the determination of cytokinin nucleotide monophosphates in coconut flesh samples, and four of them were successfully identified and quantified. The results showed that trans-zeatin riboside-5'-monophosphate was the dominant cytokinin, with a concentration of 2.7-34.2 ng/g, followed by N6-isopentenyladenosine-5'-monophosphate (≤12.9 ng/g), while the concentrations of cis-zeatin riboside-5'-monophosphate and dihydrozeatin riboside-5'-monophosphate were less than 2.2 and 4.9 ng/g, respectively.

Nonvolatile Taste Components and Antioxidant Properties of Fruiting Body and Mycelium with High Ergothioneine Content from the Culinary-Medicinal Golden Oyster Mushroom Pleurotus citrinopileatus (Agaricomycetes).

Pleurotus citrinopileatus mycelium was prepared with high ergothioneine (Hi-Ergo) content and its proximate composition, nonvolatile taste components, and antioxidant properties were studied. The ergothioneine contents of fruiting bodies and Hi-Ergo and regular mycelia were 3.89, 14.57, and 0.37 mg/g dry weight, respectively. Hi-Ergo mycelium contained more dietary fiber, soluble polysaccharides, and ash but less carbohydrates, reducing sugar, fiber, and fat than regular mycelium. However, Hi-Ergo mycelium contained the smallest amounts of total sugars and polyols (47.43 mg/g dry weight). In addition, Hi-Ergo mycelium showed the most intense umami taste. On the basis of the half-maximal effective concentration values obtained, the 70% ethanolic extract from Hi-Ergo mycelium showed the most effective antioxidant activity, reducing power, and scavenging ability, whereas the fruiting body showed the most effective antioxidant activity, chelating ability, and Trolox-equivalent antioxidant capacity. Overall, Hi-Ergo mycelium could be beneficially used as a food-flavoring material or as a nutritional supplement.

Magnesium cations assist with unpairing hydrogen-bonded 2-deoxyribose trinucleotides.

2-deoxyribose trinucleotides are essential units for storage and transfer of the genetic information. Nucleotide transpositions in trinucleotide sequences affect production of different amino acids. The study focuses on the mechanism of unpairing initially H-bonded trinucleotides. In living cells, the unpairing proceeds through DNA polymerase operating only in the presence of Mg cations. The DNA polymerase is a very complex system to be studied quantum chemically. In our simplistic approach, the polymerase is replaced by two Mg cations attached to both sides of the complementary trinucleotides. A distinguished feature of Mg in cell is in its easiness to accept and donate the electron density. In a particular molecular configuration, this makes Mg singly charged. As to the current case, we observe an unpaired electron on the Mg(+) and an unpaired electron on the trinucleotide - totally, a radical pair which coupling produces either triplet or singlet state. The study, based on the DFT B3LYP (6-311G** basis set) computations, shows that the singlet state energetically is less preferable than the triplet state. The latter is unstable and makes the trinucleotide strands unpair in the region where the singlet and triplet states cross.

2'-Alkynylnucleotides: A Sequence- and Spin Label-Flexible Strategy for EPR Spectroscopy in DNA.

Electron paramagnetic resonance (EPR) spectroscopy is a powerful method to elucidate molecular structure through the measurement of distances between conformationally well-defined spin labels. Here we report a sequence-flexible approach to the synthesis of double spin-labeled DNA duplexes, where 2'-alkynylnucleosides are incorporated at terminal and internal positions on complementary strands. Post-DNA synthesis copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions with a variety of spin labels enable the use of double electron-electron resonance experiments to measure a number of distances on the duplex, affording a high level of detailed structural information.

High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation.

Recent advances in miniaturized cell culture systems have facilitated the screening of media additives on productivity and protein quality attributes of mammalian cell cultures. However, intracellular components are not routinely measured due to the limited throughput of available analytical techniques. In this work, time profiling of intracellular nucleotides and nucleotide sugars of CHO-S cell fed-batch processes in a micro-scale bioreactor system was carried out using a recently developed high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS). Supplementation of various media additives significantly altered the intracellular nucleotides and nucleotide sugars that are inextricably linked to the process of glycosylation. The results revealed that UDP-Gal synthesis appeared to be particularly limiting whereas the impact of elevated UDP-GlcNAc and GDP-Fuc levels on the final glycosylation patterns was only marginally important. In contrast, manganese and asparagine supplementation altered the glycan profiles without affecting intracellular components. The combination of miniaturized cell cultures and high-throughput analytical techniques serves therefore as a useful tool for future quality driven media optimization studies.

Maximal dinucleotide comma-free codes.

The problem of retrieval and maintenance of the correct reading frame plays a significant role in RNA transcription. Circular codes, and especially comma-free codes, can help to understand the underlying mechanisms of error-detection in this process. In recent years much attention has been paid to the investigation of trinucleotide circular codes (see, for instance, Fimmel et al., 2014; Fimmel and Strüngmann, 2015a; Michel and Pirillo, 2012; Michel et al., 2012, 2008), while dinucleotide codes had been touched on only marginally, even though dinucleotides are associated to important biological functions. Recently, all maximal dinucleotide circular codes were classified (Fimmel et al., 2015; Michel and Pirillo, 2013). The present paper studies maximal dinucleotide comma-free codes and their close connection to maximal dinucleotide circular codes. We give a construction principle for such codes and provide a graphical representation that allows them to be visualized geometrically. Moreover, we compare the results for dinucleotide codes with the corresponding situation for trinucleotide maximal self-complementary C(3)-codes. Finally, the results obtained are discussed with respect to Crick׳s hypothesis about frame-shift-detecting codes without commas.