Base Analogues Research Articles

Local DNA Base Conformations and Ligand Intercalation in DNA Constructs Containing Optical Probes

Understanding local conformations of DNA at the level of individual nucleic acid bases and base pairs is important for elucidating molecular processes that depend on DNA sequence. Here, we apply linear absorption and circular dichroism measurements to the study of local DNA conformations, using the guanine base analog 6-methyl isoxanthopterin (6-MI) as a structural probe. We show that the spectroscopic properties of this probe can provide detailed information about the average local base and basepair conformations as a function of the surrounding DNA sequence. Based on these results we apply a simple theoretical model to calculate the circular dichroism spectra of 6-MI-substituted DNA constructs and show that our model can be used to extract information about how the local conformations of the 6-MI probe are influenced by the local base or basepair environment. We also use this probe to examine the pathway for the insertion (intercalation) of a tethered acridine ligand (9-amino-6-chloro methoxyacridine) into duplex DNA. We show that this model intercalator interacts with duplex DNA by a “displacement insertion intercalation” mechanism, whereby the acridine moiety is inserted into the DNA structure and displaces the base located opposite its attachment site. These findings suggest that site-specifically positioned base analog probes can be used to characterize the molecular and structural details of binding ligand effects on local base stacking and unstacking reactions in single- and double-stranded DNA and thus may help to define the molecular mechanisms of DNA-protein interactions that involve the site-specific intercalation of aromatic amino acid side chains into genomic DNA.

Impact of the Position of the Chemically Modified 5-Furyl-2'-Deoxyuridine Nucleoside on the Thrombin DNA Aptamer-Protein Complex: Structural Insights into Aptamer Response from MD Simulations.

Aptamers are functional nucleic acids that bind to a range of targets (small molecules, proteins or cells) with a high affinity and specificity. Chemically-modified aptamers are of interest because the incorporation of novel nucleobase components can enhance aptamer binding to target proteins, while fluorescent base analogues permit the design of functional aptasensors that signal target binding. However, since optimally modified nucleoside designs have yet to be identified, information about how to fine tune aptamer stability and target binding affinity is required. The present work uses molecular dynamics (MD) simulations to investigate modifications to the prototypical thrombin-binding aptamer (TBA), which is a 15-mer DNA sequence that folds into a G-quadruplex structure connected by two TT loops and one TGT loop. Specifically, we modeled a previously synthesized thymine (T) analog, namely 5-furyl-2′-deoxyuridine (5FurU), into each of the six aptamer locations occupied by a thymine base in the TT or TGT loops of unbound and thrombin bound TBA. This modification and aptamer combination were chosen as a proof-of-principle because previous experimental studies have shown that TBA displays emissive sensitivity to target binding based on the local environment polarity at different 5FurU modification sites. Our simulations reveal that the chemically-modified base imparts noticeable structural changes to the aptamer without affecting the global conformation. Depending on the modification site, 5FurU performance is altered due to changes in the local environment, including the modification site structural dynamics, degree of solvent exposure, stacking with neighboring bases, and interactions with thrombin. Most importantly, these changes directly correlate with the experimentally-observed differences in the stability, binding affinity and emissive response of the modified aptamers. Therefore, the computational protocols implemented in the present work can be used in subsequent studies in a predictive way to aid the fine tuning of aptamer target recognition for use as biosensors (aptasensors) and/or therapeutics.

Single-Molecule Detection of a Fluorescent Nucleobase Analogue via Multiphoton Excitation

The ability to routinely detect fluorescent nucleobase analogues at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylamine-substituted extended-6-aza-uridine (DMAthaU). We show that DMAthaU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMAthaU undergoes two-photon (2P) absorption at low excitation powers, switching to three-photon (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analogue, which facilitated the first single-molecule detection of an emissive nucleoside with multiphoton excitation.

From general base to general acid catalysis in a sodium-specific DNAzyme by a guanine-to-adenine mutation.

Recently, a few Na+-specific RNA-cleaving DNAzymes were reported, where nucleobases are likely to play critical roles in catalysis. The NaA43 and NaH1 DNAzymes share the same 16-nt Na+-binding motif, but differ in one or two nucleotides in a small catalytic loop. Nevertheless, they display an opposite pH-dependency, implicating distinct catalytic mechanisms. In this work, rational mutation studies locate a catalytic adenine residue, A22, in NaH1, while previous studies found a guanine (G23) to be important for the catalysis of NaA43. Mutation with pKa-perturbed analogs, such as 2-aminopurine (∼3.8), 2,6-diaminopurine (∼5.6) and hypoxanthine (∼8.7) affected the overall reaction rate. Therefore, we propose that the N1 position of G23 (pKa ∼6.6) in NaA43 functions as a general base, while that of A22 (pKa ∼6.3) in NaH1 as a general acid. Further experiments with base analogs and a phosphorothioate-modified substrate suggest that the exocyclic amine in A22 and both of the non-bridging oxygens at the scissile phosphate are important for catalysis for NaH1. This is an interesting example where single point mutations can change the mechanism of cleavage from general base to general acid, and it can also explain this Na+-dependent DNAzyme scaffold being sensitive to a broad range of metal ions and molecules.

The increasing impact of lethal mutagenesis of viruses.

Selection of viral mutants resistant to compounds used in therapy is a major determinant of treatment failure, a problem akin to antibiotic resistance in bacteria. In this scenario, mutagenic base and nucleoside analogs have entered the picture because they increase the mutation rate of viral populations to levels incompatible with their survival. This antiviral strategy is termed lethal mutagenesis. It has found a major impulse with the observation that some antiviral agents, which initially were considered only inhibitors of virus multiplication, may in effect exert part of their antiviral activity through mutagenesis. Here, we review the conceptual basis of lethal mutagenesis, the evidence of virus extinction through mutagenic nucleotide analogs and prospects for application in antiviral designs.

Synthesis of crystals of Tröger's base analogues and their fluorescence properties

A new strategy was developed to controllably grow the organic crystals of two Tröger's base analogues 2,8-(6H,12H-5,11-methanodibenzo[b,f]diazocineylene)-di(p-ethenylpyridine) (TBPP) and 5,6,11,12-tetrahydro-2,8-dimethylphenhomazine-di(p-ethenyl-N-pyridine) (PHZPP) from the same reactant TBPP. By adjusting the solvent conditions of the solvothermal system, TBPP crystals can be grown; on the other hand, from DMF-H2O systems with appropriate ratios, the methylene bridge of the Tröger's base would be removed and PHZPP crystalized. It is a good method to synthesize PHZPP from Tröger's base precursor under more moderate and greener solvothermal conditions. Furthermore, the different aggregation of TBPP and PHZPP crystals which may originate from the small differences in molecular structures resulted in distinct crystal fluorescent properties. The relationship between aggregate packing and photophysical properties was studied. In addition, PHZPP can be used as a pH probe for detecting H+ in solution state.

Synthesis and spectroscopic properties of axial phenoxide and para amino phenoxide incorporated indium (III) porphyrins

Tetrakis-4-methoxyphenyl indium(III) porphyrins containing phenolic and para amino phenolic moieties in the apical sites have been successfully prepared and characterized spectroscopically. The phases and structure evolutions of prepared porphyrins were deduced from powder X-ray diffraction (PXRD). The axial coordination of phenolic moieties induces slight shift of the 1H NMR signals of indium porphyrin macrocycles. The measurements of UV–Visible absorptions of porphyrins in CH2Cl2 and CH3COOC2H5 reflects that axially ligated metal chelates at neutral pH display hyperchromicity of absorption bands compared to unligated metal porphyrin. The most striking feature was the structural stability of indium porphyrins under basic (pH = 10) as well as acidic media (pH = 4). On the other hand, the metal free ligand 5, 10, 15, 20-Tetrakis-(4-methoxyphenyl) porphyrin, H2TMP (I) undergoes J type aggregation in acidic media (pH = 4). The robustness of indium porphyrins especially in acidic media are in strong contrary to other sitting atop (SAT) or out of plane (OOP) complexes which are usually demetallized under acidic pH. All the prepared macro rings exhibit negative solvatochromism reflected by the blue shift of absorption bands in CH3COOC2H5 (more polar) than in CH2Cl2 (less polar). The photoluminescence emissions in indium(III) species are significantly quenched relative to metal free ligand (I) due to internal heavy atom effect and distortion of ligand plane. The quenching of fluorescence bands are more pronounced in axially ligated metal porphyrins. The lesser excited state lifetimes of indium(III) porphyrins relative to free base analog (I) may be ascribed to spin orbit coupling induced by indium(III) ion in the respective metal chelates.

Comparative biological sensitivity and mutability of chemo-mutagens in lentil (Lens culinaris Medik)

Present investigation was carried out using three different categories of chemical mutagens viz., ethylmethane sulphonate (EMS)–an alkylating agent, hydrazine hydrate (HZ)–a base analogue and sodium azide (SA) – a respiratory inhibitor on two varieties viz., Pant L-406 and Type-8 of lentil to study the immediate biological damage induced by the mutagens and to determine the sensitivity of biological material in question. Biological damage induced in M1 generation was estimated in terms of seed germination, seedling height and pollen fertility. A dose dependent reduction with increasing concentrations of the mutagens for all these parameters was observed in both the varieties. The inhibition was more severe at the highest concentration of all the three mutagens under study. Variety Type-8 was found to be more sensitive than the var. Pant L-406 with respect to the mutagens utilized. Reduction in seed germination, seedling growth and pollen fertility in M2 generation was reasonably less as compared to M1 generation.

Synthesis of distal and proximal fleximer base analogues and evaluation in the nucleocapsid protein of HIV-1.

Anti-HIV-1 drug design has been notably challenging due to the virus' ability to mutate and develop immunity against commercially available drugs. The aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of HIV (NC). The compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. The target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against NC and HIV-1. The results of those studies are described herein.

Comparison of three-phase transformers with rectangular and circular rod contour of planar laminated magnetic cores (mass and cost indicators)

Purpose. In transformers of mass productions low power planar magnetic cores with a rectangular cross section of rods are used. With a capacity of more than 16 kV∙A and high power, transformers with circular forming loops of rods and winding coils are produced. In recent decades, in order to reduce the complexity of manufacturing, the transformers power with rectangular sections of magnetic circuits cores has increased. However, there is no analytical study of the effect of replacing circular core loops and winding coils on rectangular ones. Methodology. The influence analysis of the change in the rods configuration on the mass and cost is performed by the method of invariant and structural-parametric synthesis of electrical machines with optimized dimensionless indicators. Results. Indicators values comparison gives an objective assessment of the advantages or disadvantages of the considered variant with respect to base analog. Originality. It has been established that use of rectangular sections of rods and copper or aluminum windings forming loops in an electromagnetic system leads to an improvement in three-phase transformer mass performance of a relative to the analogue with circular generating loops. The change in mass-value indicators depends on the rods filling factor of the circular contour with rectangular packages of electrical steel. Practical value. Three-phase planar electromagnetic systems of 1–110 kV voltage class with rod cores and circular and rectangular generating circuits are distinguished by minimal mass and cost indicators by 5,76–5 % and 7,8–6,6 % and 2,5–2,3 % and 2,93–2,7 % with the rods filling factor 0,786 and 0,925 in variants with copper windings. These indicators, when used in aluminum windings, differ by 4–3,54 % and 6,3–4,9 % and by 1,8–1,75 % and 2,67–2,07 %.

Activity of base analogues (5-fluorouracil, 5-flucytosine) against planktonic cells and mature biofilm of Candida yeast. Effect of combination with folinic acid

BackgroundThe increasing number of Candida infections, especially those caused by non-C. albicans species and resistant strains, is a serious medical problem. ObjectivesIn this study, the antifungal activity of base analogues, 5-flucytosine (5-FC) and 5-fluorouracil (5-FU), was tested against planktonic cells as well as against mature biofilm. MethodsTests were performed according the EUCAST methodology. Antibiofilm effectiveness of tested drugs was determined by the crystal violet staining method. The cytotoxicity assays was performed according to the ISO 10993-5 norm. Results5-FC and 5-FU were effective against fifteen fluconazole resistant Candida glabrata strains with an average minimal inhibitory concentration (MIC) of 0.152mg/L and 0.39mg/L, respectively. Folinic acid (folinate- e.g., leucovorin) is a common drug used in oncology simultaneously with 5-FU. In our tests folinate was able to lower MIC for 5-FC from 0.152 to 0.058mg/L (P<0.05). In the biofilm assay 5-FU and 5-FC alone did not induce any changes in the biomass of mature biofilm. Addition of folinate to each base analogue resulted in up to 90% reduction of biomass. Viability tests show that a concentration of 64mg/L of 5-FC and 5-FU supplemented with folinate can be fungicidal against mature biofilms of some Candida isolates. No cytotoxic effect was found for combination of FOL and 5-FC. ConclusionTherapy of 5-FU+folinate is well known in cancer treatment, in this study we reveal the beneficial effect of folinate on antifungal activity of 5-FC as well as the antifungal potential of 5-FU+folinate.

Undergraduate Student (Mis)understandings of Mutations

Genetics is known to have difficult concepts to learn and understand due to its use of complex, nuanced, and technical vocabulary, as well as the abstract nature of many concepts, the requirement to think across multiple scales, particularly the symbolic scale, and the pace of change to the field. The concept of mutation and its effects are particularly important due to the foundational level they play in many critical areas of modern biology including evolution, central dogma, genotype‐phenotype connections, genomic identity, and personalized medicine. This project aimed to gain a thorough perspective of student understanding and alternative concepts they hold regarding mutations. Student response data were gathered using open‐ended questions focused on three distinct learning objectives. The student should be able to: 1) define mutation, 2) categorize changes to DNA and predict the outcome of these changes on a protein produced from the altered DNA using the genetic code, 3) differentiate between somatic and germline mutations and predict the inheritance patterns of each type of mutation, 4) predict the nature of changes to DNA exposed to intercalating agents, base analogs, and radiation (ionizing and non‐ionizing). More than 400 student responses to seventeen questions were analyzed qualitatively. Student responses showed that students have major issues understanding: 1) the difference between a mutation and any change in RNA, protein, or function, 2) central dogma terminology and how genetic information “flows” within it, 3) the relationships of somatic and germline mutations to heritability, and 4) how mutagens cause mutations. It is hoped that knowing which alternative conceptions students commonly hold will aid faculty in designing instruction that enables students to form a more accurate conceptual framework regarding mutation.Support or Funding InformationNational Science Foundation Grant 1710262 to RLST and NMBThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Antibiotic Trimethoprim Displays Strong Mutagenic Synergy with 2-Aminopurine.

We show that trimethoprim (TMP), an antibiotic in current use, displays a strong synergistic effect on mutagenesis in Escherichia coli when paired with the base analog 2-aminopurine (2AP), resulting in a 35-fold increase in mutation frequencies in the rpoB-Rifr system. Combination therapies are often employed both as antibiotic treatments and in cancer chemotherapy. However, mutagenic effects of these combinations are rarely examined. An analysis of the mutational spectra of TMP, 2AP, and their combination indicates that together they trigger a response via an alteration in deoxynucleoside triphosphate (dNTP) ratios that neither compound alone can trigger. A similar, although less strong, response is seen with the frameshift mutagen ICR191 and 2AP. These results underscore the need for testing the effects on mutagenesis of combinations of antibiotics and chemotherapeutics.

Crystallographic elucidations of indium(III) porphyrin conformations, morphology and aggregation behaviour: Comparative optical study of free base porphyrins and their indium(III) derivatives at varying pH

The supramolecular structural architectures of 5, 10, 15, 20-Tetrakis-(4-methoxyphenylporphyrinato) indium (III) chloride (I) and 5, 10, 15, 20-Tetrakis-(4-methylphenylporphyrinato) indium (III) chloride (II) have been determined from their three dimensional X-ray diffraction data. Complex I crystallizes in the orthorhombic space group, Pbca with a = 23.0083 (4) Å, b = 15.2447 (3) Å, c = 23.3737 (4) Å, α = β = γ = 90°, V = 8198.5 (3) Å3 and Z = 8, whereas complex II crystallizes in the triclinic space group, P-1 with a = 12.0737 (5) Å, b = 12.1000 (5) Å, c = 14.1682 (6) Å, α = 88.560 (3)°, β = 78.730 (3)°, γ = 78.280 (3)° V = 1987.40 (14) Å3 and Z = 2. The geometries around In+3 ions in both the complexes are square pyramidal giving dome shaped architectures to the metalloporphyrins. In their respective crystal lattices, the monomeric units of complex I and II are packed in J-type (head-to-tail) supramolecular aggregation. These monomers are linked into their three dimensional supramolecular networks by various non covalent forces. Further the packing of monomers of each complex in their respective unit cells leaves significant void volumes (porosities). The optical absorption spectra of complex I and II and their free base analogues i.e. 5, 10, 15, 20-Tetrakis-(4-methoxyphenyl) porphyrin, H2TMP and 5, 10, 15, 20-Tetrakis-(4-methylphenyl) porphyrin, H2TTP have also been investigated in CH2Cl2 at different ionic strengths of the media. The investigations revealed that both the free bases exist as simple monomers, j-aggregates and dianionic monomers at neutral (pH = 7), acidic (pH = 4 and lower) and basic (pH = 10) media conditions. Also at high ionic strength of the media, the free bases undergo charge transfer electronic transitions. The two In3+ porphyrin exists as simple monomers at pH = 7. However at pH = 5 and lower, the In3+ ions in complex II is remarkably easily replaced by the protons of the acid to generate the corresponding protonated free base monomers which simultaneously aggregate in J-type stacking mode. In contrast to complex II that exhibits low stability in acidic media, the complex I remain intact in such media reflecting extremely high stability of the complex. The insertion of In3+ ions into the porphyrinic cores of free bases causes bathochromic shift followed by increase in the intensities of absorption bands. The appearance of two set of doublets for ortho phenyl protons in the 1H NMR spectra of each complex is attributed to the magnetic inequivalence of their ortho phenyl protons caused by the pulling of In+3 ions above the mean porphyrin planes.

Optically active and photoswitchable Tröger's base analogs

Azo carrying Tröger's base analogs introduced chirality, thermochromic and phototunable properties to an achiral liquid crystal host.

A Study on the Methods of Facilitating Evaluation of a Base Analog in Analogical Reasoning: Focusing on the Viewpoint of Similarity between Base and Target

A Study on the Methods of Facilitating Evaluation of a Base Analog in Analogical Reasoning: Focusing on the Viewpoint of Similarity between Base and Target

Synthesis and characterization of Schiff base analogues of fluoroaniline and their antibiocidal activity against MRSA

Synthesis and characterization of Schiff base analogues of fluoroaniline and their antibiocidal activity against MRSA

Computational design and characterization of new thieno‐expanded tricyclic purine analogs

AbstractUnnatural nucleobases are under intense research due to their widespread applications in nucleic acids research. In this work, four new thieno‐expanded purine analogs comprising ttzA, tthA, ttzG, and tthG were computationally designed based on the isomorphic tz‐ and th‐bases. These base analogs can also be seen as modified derivatives of the previously reported tricyclic purine analogs (ttA and ttG). The structural, electronic, and photophysical properties are studied by means of DFT and TDDFT calculations. We find out that these new bases can form stable Watson‐Crick base pairs with natural counterparts, thus potentially mimicking natural nucleobases in DNA/RNA duplexes. Calculations reveal that these bases have smaller AIPs and HOMO‐LUMO gaps than natural ones, suggesting that they are candidates for applications in nanowire technology. Particularly, the photophysical properties were explored, and the results are compared with those for tz‐, th‐, and tt‐bases. The nature of the low‐lying excited states is discussed, and analyses reveal that the thiophene‐homologation would not change excitation maxima of thA and tzA, while it will result in large red‐shifts of those of thG and tzG. Meanwhile, thiophene insertion has relatively larger influences on the emissions thA and tzG, for which the fluorescence was 37 nm blue‐shifted and 19 nm red‐shifted, respectively. Taking these new bases as derivatives of ttA and ttG, it was found that the modifications would result in large red‐shifts of both the excitation maxima and the fluorescence. The effects of water solution and base paring on the phtotophysical properties were also considered.

EGFR Tyrosine Kinase Targeted In Silico Design and Synthesis of Novel Quinazoline Derivatives

Quinazolines are medicinally important as anti-convulsant, anti-cancer, anti-microbial, and anti-tubercular properties etc. They target epidermal growth factor receptors on tumor cells. This work is aimed to synthesize a new series of quinazoline derivatives which could deliver drug specifically to the EGFR over expressing tumors. A series of novel Schiff’s base analogues were developed by in silico screening methods. The drug likeness of the analogues was analyzed by using Molinspiration software. Biological activities of these analogues were evaluated by using PASS software. The candidates which obeyed Lipinski rule of five and having suitable anti-cancer and anti-microbial activity were taken for docking studies using Schrodinger software. All the proposed derivatives were docked with various protein targets obtained from PDB, using GLIDE software and satisfactory docking energy scores were obtained. Selected 10 derivatives of quinazoline have been synthesized by anthranilamide and benzaldehyde as starting materials. These analogues were purified by analyzing its melting point, Rf value. These were further characterized by FT-IR, 1H NMR and MASS spectral studies. The anti-cancer activity of these derivatives was done by MTT assay against HeLa cell lines, LD50 values were calculated by Brine Shrimp Lethality Assay. From these experiments it is clearly revealed that these analogues possess good anti-cancer activity and are good lead compounds against tumors.

Daniel McGillivray Brown. 3 February 1923—24 April 2012

Dan Brown was a nucleic acids chemist of the highest order, beginning with pioneering work under Lord Alexander Todd in the 1950s at University of Cambridge on chemical methods for synthesis of nucleosides and nucleotides. This work helped to confirm the furanose chemical structure of the sugar in nucleosides as well as the 3′-5′ phosphodiester linkage in DNA and RNA, perhaps the most well thought of achievement of his career. Later, as a chemistry department lecturer, he established the chemical structures of glycerol monophosphoinositides as well as triphosphoinositides. Turning back to the nucleic acids in 1961, he became fascinated by the effect of mutagens on DNA. He elucidated the mechanism for the reaction of hydroxylamine on cytidine to form an initial ‘bis-adduct’ and thereafter N 6 -hydroxycytidine. Moving in 1982 to the MRC Laboratory of Molecular Biology, he developed a method to prepare single-stranded DNA probes for detection of RNA sequences and in addition worked on a novel automated device for oligonucleotide synthesis. Reverting to his interest in mutagens, he then designed and synthesized hydrogen bonding degenerate bases and developed novel P and K modified pyrimidine and purine bases respectively as transition mutagens. Finally, he synthesized the base analogue 5-nitroindole as a potential universal base, which became useful in cycle DNA sequencing, and in addition developed the concept of ‘error catastrophe’ for the ribonucleoside of the P base as an antiviral agent. The P, K and 5-nitroindole bases became the most valued chemical entities of his career to molecular biologists. His legacy to the nucleic acids includes both his significant contributions to studies of the chemical nature of DNA and RNA and their constituents as well as a variety of enabling nucleic acids chemistry methods and mechanisms of DNA mutagenicity.