Base Analogues Research Articles

The Pivotal Role of the Mitochondrial Amidoxime Reducing Component 2 in Protecting Human Cells against Apoptotic Effects of the Base Analog N6-Hydroxylaminopurine

N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.

DNA polymerase-mediated synthesis of unbiased threose nucleic acid (TNA) polymers requires 7-deazaguanine to suppress G:G mispairing during TNA transcription.

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9°N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA. However, our ability to transcribe four-nucleotide libraries has been limited by chain termination events that prevent the synthesis of full-length TNA products. Here, we show that chain termination is caused by tG:dG mispairing in the enzyme active site. We demonstrate that the unnatural base analogue 7-deazaguanine (7dG) will suppress tGTP misincorporation by inhibiting the formation of Hoogsteen tG:dG base pairs. DNA templates that contain 7dG in place of natural dG residues replicate with high efficiency and >99% overall fidelity. Pre-steady-state kinetic measurements indicate that the rate of tCTP incorporation is 5-fold higher opposite 7dG than dG and only slightly lower than dCTP incorporation opposite either 7dG or dG. These results provide a chemical solution to the problem of how to synthesize large, unbiased pools of TNA molecules by polymerase-mediated synthesis.

Syntheses, crystal structure, Hirshfeld surfaces, fluorescence properties, and DFT analysis of benzoic acid hydrazone Schiff bases

Two hydrazone Schiff base analogues, namely, (E)-N′-(4-hydroxy-3-methoxybenzylidene)benzohydrazide (3a) and (E)-N′-(4-methoxybenzylidene)benzohydrazide (3b), were synthesized using a mild, efficient method and characterized by 1H NMR, mass spectrometry, elemental analysis, and single-crystal X-ray diffraction. X-ray analysis of a single crystal of 3a revealed a tetragonal, space group I4(1)/a structure, with an E-configuration around the azomethine (C8N2) double bond. In this structure, the NH and OH groups act as proton donors and the >CO and N groups as proton acceptors, and these facilitate hydrogen bond formation in the crystal state. Plausible intermolecular interactions were studied using 3D Hirshfeld surfaces and related 2D fingerprint plots. The optimized geometry, vibrational frequencies, Mulliken charge distribution, molecular electrostatic potential (MEP) maps, frontier molecular orbitals (FMOs), and associated energies of the ground state and the first single excited state were calculated using density functional theory (DFT) and time-dependant DFT calculations using the B3LYP/6-311G method. Vibrational frequencies calculated in the gaseous phase compared with experimental values measured in the solid state and showed good agreement with each other. The chemical reactivities of 3a and 3b were predicted by mapping MEP surface over optimized geometries and comparing these with MEP map generated over crystal structures. Mulliken charge distribution analysis and MEP map of 3a and 3b revealed that N(1), O(1), O(2) and O(3) atoms could act as electron donors and coordinate with metals and that these represented the most suitable sites for electrophilic attack. In fluorescence spectra, the absorption and emission spectra of 3a and 3b were similar in different polar solvents with few exceptions. In addition, both compounds exhibited dual emission spectra in acetone due to keto-enol tautomerism induced by photoexcitation.

Second-generation fluorescent quadracyclic adenine analogues: environment-responsive probes with enhanced brightness.

Fluorescent base analogues comprise a group of increasingly important molecules for the investigation of nucleic acid structure, dynamics, and interactions with other molecules. Herein, we report on the quantum chemical calculation aided design, synthesis, and characterization of four new putative quadracyclic adenine analogues. The compounds were efficiently synthesized from a common intermediate through a two-step pathway with the Suzuki-Miyaura coupling as the key step. Two of the compounds, qAN1 and qAN4, display brightnesses (εΦF) of 1700 and 2300, respectively, in water and behave as wavelength-ratiometric pH probes under acidic conditions. The other two, qAN2 and qAN3, display lower brightnesses but exhibit polarity-sensitive dual-band emissions that could prove useful to investigate DNA structural changes induced by DNA-protein or -drug interactions. The four qANs are very promising microenvironment-sensitive fluorescent adenine analogues that display considerable brightness for such compounds.

New size-expanded RNA nucleobase analogs: A detailed theoretical study

Fluorescent nucleobase analogs have attracted much attention in recent years due to their potential applications in nucleic acids research. In this work, four new size-expanded RNA base analogs were computationally designed and their structural, electronic, and optical properties are investigated by means of DFT calculations. The results indicate that these analogs can form stable Watson–Crick base pairs with natural counterparts and they have smaller ionization potentials and HOMO–LUMO gaps than natural ones. Particularly, the electronic absorption spectra and fluorescent emission spectra are calculated. The calculated excitation maxima are greatly red-shifted compared with their parental and natural bases, allowing them to be selectively excited. In gas phase, the fluorescence from them would be expected to occur around 526, 489, 510, and 462nm, respectively. The influences of water solution and base pairing on the relevant absorption spectra of these base analogs are also examined.

Monitoring translocation of multisubunit RNA polymerase along the DNA with fluorescent base analogues.

Here we describe a direct fluorescence method that reports real-time occupancies of the pre- and post-translocated state of multisubunit RNA polymerase. In a stopped-flow setup, this method is capable of resolving a single base-pair translocation motion of RNA polymerase in real time. In a conventional spectrofluorometer, this method can be employed for studies of the time-averaged distribution of RNA polymerase on the DNA template. This method utilizes commercially available base analogue fluorophores integrated into template DNA strand in place of natural bases. We describe two template DNA strand designs where translocation of RNA polymerase from a pre-translocation to a post-translocation state results in disruption of stacking interactions of fluorophore with neighboring bases, with a concomitant large increase in fluorescence intensity.

Mechanistic Studies of DNA-Protein Interactions in Bacteriophage T4 DNA Replication Complexes at Single-Base Resolution

Combining biophysical measurements on the function and control of T4 bacteriophage replication complexes with detailed structural information can throw light onto the mechanism of action of these ‘macromolecular machines’. In this study we use the low energy circular dichroism and fluorescent properties of site-specifically introduced fluorescent base analogues to monitor the binding and interactions of gene 32 protein (gp32), the ssDNA binding protein of the T4 DNA replication complex, and gene 59 protein (gp59), the helicase loader protein involved in loading the T4 primosome complex onto replication forks. We show that gp32 binds preferentially to the 3'-end of a short ssDNA oligomer and that only two or three nucleotide bases at the 3' end are directly perturbed by the binding interaction, although the binding site size in the polynucleotide binding mode is 7 nts. Loss of exciton coupling in the CD spectrum and an increase in fluorescence intensity shows that gp32 binding causes extension of the sugar-phosphate backbone of the oligonucleotide. Mechanisms involved in gp32 binding to its ssDNA targets in both the isolated and the cooperative binding modes will be described. In addition our CD and fluorescence experiments with base analogue probes show the gp59 binding to a forked DNA construct perturbs the bases at the junction and suggests that once gp41 is loaded the perturbation extends deep into the double helix. Mechanisms whereby these proteins work in tandem in setting up various functional interactions with the other components of the DNA replication complex will be discussed.

Development of a visible nanothermometer with a highly emissive 2′-O-methylated guanosine analogue

We have synthesized a fluorescent base analogue, 2-aminothieno[3,4-d]pyrimidine based G-mimic deoxyribonucleoside, 2′-OMe-thG, and investigated its photophysical properties and DNA incorporation.

Stacking of the mutagenic base analogue 5-bromouracil: energy landscapes of pyrimidine dimers in gas phase and water.

The potential energy surfaces of stacked base pairs consisting of cytosine (C), thymine (T), uracil (U) and the mutagenic thymine analogue 5-bromouracil (BrU) have been searched to obtain all possible minima. Minima and transition states were optimised at the counterpoise-corrected M06-2X/6-31+G(d) level, both in the gas phase and in water, modelled by the polarizable continuum model. The stacked dimers studied are BrU/BrU, C/BrU, C/C, C/T, C/U, T/BrU and T/U. Both face-to-back and face-to-face structures were considered. Free energies were calculated at 298.15 K. Together with U/U, T/T and BrU/U results from previous work, these results complete the family consisting of every stacked dimer combination consisting of C, T, U and BrU. The results were used to assess the hypothesis suggested in the literature that BrU stacks stronger than T, which could stabilise the mispair formed by BrU and guanine. In the gas phase, structures of C/BrU, T/BrU and U/BrU with greater zero-point-corrected binding energies than C/T, T/T and U/T, respectively, were found, with differences in favour of BrU of 3.1 kcal mol(-1), 1.7 kcal mol(-1) and 0.5 kcal mol(-1), respectively. However, the structure of these dimers differed considerably from anything encountered in DNA. When only the dimers with the most "DNA-like" twist (±36°) were considered, C/BrU and T/BrU were still more strongly bound than C/T and T/T, by 0.5 kcal mol(-1) and 1.7 kcal mol(-1), respectively. However, when enthalpic and/or solvent contributions were taken into account, the stacking advantage of BrU was reversed in the gas phase and mostly nullified in water. Enhanced stacking therefore does not seem a plausible mechanism for the considerably greater ability of BrU-G mispairs over T-G mispairs to escape enzymatic repair.

Effect of mismatch repair on the mutation rate of bacteriophage ϕX174.

Viral mutation rates vary widely in nature, yet the mechanistic and evolutionary determinants of this variability remain unclear. Small DNA viruses mutate orders of magnitude faster than their hosts despite using host-encoded polymerases for replication, which suggests these viruses may avoid post-replicative repair. Supporting this, the genome of bacteriophage ϕX174 is completely devoid of GATC sequence motifs, which are required for methyl-directed mismatch repair in Escherichia coli. Here, we show that restoration of the randomly expected number of GATC sites leads to an eightfold reduction in the rate of spontaneous mutation of the phage, without severely impairing its replicative capacity over the short term. However, the efficacy of mismatch repair in the presence of GATC sites is limited by inefficient methylation of the viral DNA. Therefore, both GATC avoidance and DNA under-methylation elevate the mutation rate of the phage relative to that of the host. We also found that the effects of GATC sites on the phage mutation rate vary extensively depending on their specific location within the phage genome. Finally, the mutation rate reduction afforded by GATC sites is fully reverted under stress conditions, which up-regulate repair pathways and expression of error-prone host polymerases such as heat and treatment with the base analog 5-fluorouracil, suggesting that access to repair renders the phage sensitive to stress-induced mutagenesis.

Local and Global Folding in a 58mer RNA Revealed by 2-Aminopurine Substitutions and Specific NMR Labels

The ability of RNA to sample multiple conformations allows it to fulfill a myriad of biological roles. The folding pathways that RNA takes to achieve these conformations are not intuitive, and there is a pressing need to predictively model this folding behavior. The GTPase center (GAC) of the large subunit of the prokaryotic ribosome, is a well-studied 58mer that exhibits ion-dependent tertiary folding. By substituting the fluorescent base analog 2-aminopurine (2AP), 15N labeled bases, or 13C labeled bases in to specific loop and bulge regions we are able to monitor local changes that occur during Mg2+ induced tertiary folding. Being able to contrast the results from stopped flow kinetics, NMR dynamics and structural studies, we can see how local interactions make up tertiary folding. This work was funded by the NIH R01-GM098102 to KBH. Labeled RNA molecules were contributed by Agilent and NMR studies were supported by Agilent.

Explaining the abundance of distant analogies in naturalistic observations of experts.

Analogical reasoning is a landmark of human cognition. Based on the realization that the elements of two situations are organized by similar systems of relations, analogical inferences allow the transfer of knowledge structures from a better-known situation (the base analog) to a target situation that is relatively less understood (the target analog). Experimental research has demonstrated that the retrieval of base analogs from long term memory in response to the proceesing of a target analog is infrequent in the lack of semantic similarities between both situations (Gick and Holyoak, 1980; Keane, 1987; Gentner et al., 1993; Trench and Minervino, 2014). With the turn of the century, several naturalistic observations of experts working in their domains of expertise yielded a more complex picture. While molecular biologists (Dunbar, 1997) and psychologists (Saner and Schunn, 1999) still exhibited mostly within-domain analogizing, the observation of journalists and politicians (Blanchette and Dunbar, 2001), teachers (Richland et al., 2004), managers (Bearman et al., 2007) and design engineers (Christensen and Schunn, 2007) showed a more frequent use of long-distance analogies. The naturalistic study by Kretz and Krawczyk (2014) on the use of analogies by economists also demonstrates an abundance of distant analogies in the sevice of an impressive variety of communicative purposes, most of which were not evident in prior research. These goals included the generation of concrete source examples of more general target concepts, the formation of visual images of source concepts, the addition of colorful speech, the inclusion of a target into a source concept, or the differentiation between source and target concepts. With these results in mind, the time is ripe to assert that the naturalistic observation of experts shows a more flexible use of analogical sources than is predicted by experimental studies on analogical transfer, and simulated by dominant computer models of analogical retrieval (e.g., MAC/FAC, Forbus et al., 1995; LISA, Hummel and Holyoak, 1997). How, then, to explain this analogical abundance? In trying to account for the contrasting results of the experimental and the naturalistic traditions, the default explanations revolve around the expertise of the analogizers and the psychological constraints of the target tasks. I will argue that although both factors are likely to bear some responsibility for this empirical inconsistency, there are reasons to expect a heavier weight of the latter.

A 28 Gb/s 560 mW Multi-Standard SerDes With Single-Stage Analog Front-End and 14-Tap Decision Feedback Equalizer in 28 nm CMOS

This paper presents a 28 Gb/s multistandard SerDes macro which is fabricated in TSMC 28 nm CMOS process. The transimpedance amplifier (TIA) base analog front-end achieved 15 dB high-frequency boost with an on-chip compact passive inductor. The adaptation loop for the boost is decoupled from the decision feedback equalizer (DFE) adaptation by the use of a group delay algorithm. The DFE is a half-rate 1-tap unrolled design with only two total error latches for power and area reduction. A two-stage sense amplifier-based latch achieved sensitivity of 15 mV. The high-speed clock buffer uses a PMOS active inductor circuit with common-mode feedback to optimize the circuit performance. The transceiver achieves error-free operation at 28 Gbps with 34 dB channel loss, consumes the worst case power of 560 mW/lane, and fully complies with multiple standards and applications.

Synthesis, antimicrobial and anti-biofilm activities of novel Schiff base analogues derived from methyl-12-aminooctadec-9-enoate

A novel library of Schiff base analogues (5a–q) were synthesized by the condensation of methyl-12-aminooctadec-9-enoate and different substituted aromatic aldehydes. The synthesized compounds were thoroughly characterized by spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, ESI-MS and HRMS). The Schiff base analogues with different substitutions were screened for in vitro antibacterial activity against 7 different bacterial strains. Among these, the compounds with electron withdrawing substituent, namely chlorine (5a) and electron donating substituents, namely hydroxy (5n) and methoxy (5o), were found to exhibit excellent to good antimicrobial activities (MIC value 9–18μM) against Staphylococcus aureus MTCC 96, Staphylococcus aureus MLS-16 MTCC 2940 and Bacillus subtilis MTCC 121. The products were also screened for anti-biofilm and MBC (Minimum Bactericidal Concentration) activities which exhibited promising activities.

Antibacterial and cytotoxic activities of Schiff base analogues of 4-aminoantipyrine

Schiff base is the class of compounds showing wide range of biological activities having the azomethine (-N=CH-) active pharmacophore, which play major roles in their significant bio-activities. A series of Schiff base analogues of 4-aminoantipyrine analogues have been tested for bactericidal and cytotoxic activities against selected bacterial strains and brine shrimp (Artemia salina) nauplii, respectively. Of the compounds tested, two compounds showed a good inhibition of bacterial growth against E. coli and C. sakazakii, whereas three compounds demonstrated high cytotoxicity with LC50 values of 225, 480, and 581 ppm, in a short term bioassay using A. salina. Qualitative structure-cytotoxic activity relationships were studied using physicochemical parameters; a good correlation between clogP and cytotoxic activity was observed.

Correction to “Control of the Stability of a Protein–RNA Complex by the Position of Fluorine in a Base Analogue”

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionCorrection to “Control of the Stability of a Protein–RNA Complex by the Position of Fluorine in a Base Analogue”Yulia Benitex and Anne M. Baranger*Cite this: J. Am. Chem. Soc. 2014, 136, 35, 12514Publication Date (Web):August 22, 2014Publication History Published online22 August 2014Published inissue 3 September 2014https://doi.org/10.1021/ja507210hCopyright © 2014 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views556Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (114 KB) Get e-Alerts Get e-Alerts

Discovery of Tröger's base analogues as selective inhibitors against human breast cancer cell line: Design, synthesis and cytotoxic evaluation

A library of structurally diverse Tröger's base analogues has been constructed via unusual amination of methylene bridge employing Vilsmeier–Haack conditions as well as by the incorporation of five and six membered heterocycles on the aromatic core of Tröger's base framework. The constructed structurally diverse frameworks were evaluated for their cytotoxic activities against a panel of three human cancer lines A549 (lung adenocarcinoma), MDAMB-231 (breast) and SK-N-SH (neuroblastoma). From the activity profile obtained, a redesign of Tröger's base analogues led to the construction of more potent molecular entities. The study led to development of a series of compounds with MDAMB-231 cell line specific cytotoxicity. Of the 30 compounds synthesized and evaluated, 7 compounds were found to possess cytotoxicity that is equivalent or better than standard drug doxorubicin against MDAMB-231 cell line while only one compound was found to be active against SK-N-SH cell line.

Molecular cloning and expression of high GC-rich novel tumor suppressor gene HIC-1.

Hypermethylated in Cancer-1 (HIC-1) is a novel tumor suppressor plays crucial role in tumor formation through loss of function by hypermethylation. HIC-1 is known as transcriptional factor whereas little known about its structure and function. Requirement felt to clone and express full coding protein and reveal various domains and binding pattern onto promoters conducting biophysical studies which lack in current scenario. Production of sufficient amounts of protein is frequent bottleneck in structural biology. Cloning full-length HIC-1 with >73 % GC content poses a daunting task with sequencing and expression adds more to the challenge. We describe the methodology for specific amplification, cloning, sequencing, and expression of HIC-1 in E. coli. Standardization using 1.5 U pfu polymerase in (NH4)2SO4 containing buffer gave specific amplification with 10 % DMSO and 1.5 mM MgCl2. Sequencing achieved using base analog 7-de aza dGTP (0.2 mM) or denaturant like DMSO (10 %) or betaine (1 M). Expression using strains of E. coli induced by different concentrations of IPTG (0.5-5.0 mM) for time points of 4, 8, 16, 20, and 24 h at different temperatures 25, 30, and 37 °C. Full-length clone successfully expressed in BL21-Codon Plus-RP using 1 mM concentration of IPTG for 8 h at 37 °C gave prominent band of 74 kDa.

Synthesis of enantiomerically pure Tröger's base derivatives via chiral disulfoxides.

The preparation, full characterization and determination of the absolute configuration of diastereomerically pure disulfoxide derivatives of Tröger's base are described. A sulfinyl-lithium exchange followed by the reaction with electrophiles affords synthetically meaningful amounts of enantiomerically pure 4,10-disubstituted Tröger's base analogues without any erosion of the stereogenic information at the nitrogen centers.

Mechanical Properties of Base-Modified DNA Are Not Strictly Determined by Base Stacking or Electrostatic Interactions

This work probes the mystery of what balance of forces creates the extraordinary mechanical stiffness of DNA to bending and twisting. Here we explore the relationship between base stacking, functional group occupancy of the DNA minor and major grooves, and DNA mechanical properties. We study double-helical DNA molecules substituting either inosine for guanosine or 2,6-diaminopurine for adenine. These DNA variants, respectively, remove or add an amino group from the DNA minor groove, with corresponding changes in hydrogen-bonding and base stacking energy. Using the techniques of ligase-catalyzed cyclization kinetics, atomic force microscopy, and force spectroscopy with optical tweezers, we show that these DNA variants have bending persistence lengths within the range of values reported for sequence-dependent variation of the natural DNA bases. Comparison with seven additional DNA variants that modify the DNA major groove reveals that DNA bending stiffness is not correlated with base stacking energy or groove occupancy. Data from circular dichroism spectroscopy indicate that base analog substitution can alter DNA helical geometry, suggesting a complex relationship among base stacking, groove occupancy, helical structure, and DNA bend stiffness.