DNA Building Blocks Research Articles

Modelling charge transfer processes in C2+-tetrahydrofuran collision for ion-induced radiation damage in DNA building blocks.

Investigations of collision-induced processes involving carbon ions and molecules of biological interest, in particular DNA building blocks, are crucial to model the effect of radiation on cells in order to improve medical treatments for cancer therapy. Using carbon ions appears to be one of the most efficient ways to increase biological effectiveness to damage cancerous cells by irradiating deep-seated tumors. Therefore, interest in accurate calculations to understand fundamental processes occurring in ion-molecule collision systems has been growing recently. In this context, the charge transfer process in the collision of C2+(1s22s2) ions with the heterocyclic sugar moiety building block tetrahydrofuran (THF) was studied in order to interpret the mechanisms occurring at the molecular level. The molecular structure properties of THF were obtained by means of ab initio quantum chemistry methods. The role of the conformational structure and the orientation of the THF molecule in collision with C2+ ions are particularly discussed. Anisotropic effects of the process dynamics in the collision energy ranging from eV to keV by means of semiclassical treatment are also presented and compared to previous experimental and theoretical investigations. A detailed analysis of the obtained cross sections points out an increase in these values by three orders of magnitude by a change of the THF symmetry from C2v to Cs in collision with C2+, which determines a more efficient charge transfer in this case.

Resolving the Origins of Crystalline Anharmonicity Using Terahertz Time-Domain Spectroscopy and ab Initio Simulations.

Anharmonicity has been shown to be an important piece of the fundamental framework that dictates numerous observable phenomena. In particular, anharmonicity is the driving force of vibrational relaxation processes, mechanisms that are integral to the proper function of numerous chemical processes. However, elucidating its origins has proven difficult due to experimental and theoretical challenges, specifically related to separating the anharmonic contributions from other unrelated effects. While no one technique is particularly suited for providing a complete picture of anharmonicity, by combining multiple complementary methods such a characterization can be made. In this study the role of individual atomic interactions on the anharmonic properties of crystalline purine, the building block of many DNA and RNA nucleobases, is studied by experimental terahertz time-domain spectroscopy and first-principles density functional theory (DFT) and ab initio molecular dynamics simulations (AIMD). In particular, the detailed vibrational information provided by the DFT calculations is used to interpret the atomic origins of anharmonic-related effects as determined by the AIMD calculations, which are in good agreement with the experimental data. The results highlight that anharmonicity is especially pronounced in the intermolecular interactions, particularly along the amine hydrogen bond coordinate, and yields valuable insight into what is similarly observed complex biosystems and crystalline solids.

Electrical detection and quantification of single and mixed DNA nucleotides in suspension.

High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution.

Self-Assembled DNA Hydrogel Based on Enzymatically Polymerized DNA for Protein Encapsulation and Enzyme/DNAzyme Hybrid Cascade Reaction.

DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system.

N,N,O and N,O,N Meridional cis Coordination of Two Guanines to Copper(II) by d(CGCGCG)2.

Many research groups study the generation of supramolecular n-dimensional arrays by combining metals with DNA building blocks. Most of the time, the natural nucleobases are modified to obtain higher-affinity metal binding sites. Using unmodified nucleobases avoids a potentially difficult synthesis; however, they have the possible disadvantage of a less defined and/or weaker coordination mode of the metal. Structural studies on the behavior of copper(II) as a linking metal and guanine as the natural ligand for metals in unmodified DNA are reported. Previously, the ability of mono- and dinuclear metal complexes to induce Z-DNA has been explored [Medina-Molner, A.; Spingler, B. Chem. Commun. 2012, 48, 1961; Medina-Molner, A.; Rohner, M.; Pandiarajan, D.; Spingler, B. Dalton Trans. 2015, 44, 3664]. Herein, X-ray crystallographic studies of the structures resulting from the combination of copper(II) ions with DNA hexamers of the general sequence d(CG)3 are presented. Three different packing motifs were observed in three crystal structures with resolutions ranging from 2.15 to 1.45 Å. The motifs are dependent upon other cations being present and/or the crystallization conditions. The first examples of intramolecular O6,N7-chelates of a neutral purine nucleobase to copper(II) were obtained as well as the first meridional N,N,O and N,O,N coordination modes of two guanines to copper(II). The fascinating coordination chemistry of copper(II) complexes generated by the Z-DNA oligonucleotides and the differences to simple nucleobases complexes with copper(II) are discussed in detail.

Flavin-Dependent Thymidylate Synthase as a New Antibiotic Target.

In humans de novo synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

Gas-Phase Femtosecond Particle Spectroscopy: A Bottom-Up Approach to Nucleotide Dynamics.

We summarize how gas-phase ultrafast charged-particle spectroscopy has been used to provide an understanding of the photophysics of DNA building blocks. We focus on adenine and discuss how, following UV excitation, specific interactions determine the fates of its excited states. The dynamics can be probed using a systematic bottom-up approach that provides control over these interactions and that allows ever-larger complexes to be studied. Starting from a chromophore in adenine, the excited state decay mechanisms of adenine and chemically substituted or clustered adenine are considered and then extended to adenosine mono-, di-, and trinucleotides. We show that the gas-phase approach can offer exquisite insight into the dynamics observed in aqueous solution, but we also highlight stark differences. An outlook is provided that discusses some of the most promising developments in this bottom-up approach.

Spatial colocalization and functional link of purinosomes with mitochondria

Purine biosynthetic enzymes organize into dynamic cellular bodies called purinosomes. Little is known about the spatiotemporal control of these structures. Using super-resolution microscopy, we demonstrated that purinosomes colocalized with mitochondria, and these results were supported by isolation of purinosome enzymes with mitochondria. Moreover, the number of purinosome-containing cells responded to dysregulation of mitochondrial function and metabolism. To explore the role of intracellular signaling, we performed a kinome screen using a label-free assay and found that mechanistic target of rapamycin (mTOR) influenced purinosome assembly. mTOR inhibition reduced purinosome-mitochondria colocalization and suppressed purinosome formation stimulated by mitochondria dysregulation. Collectively, our data suggest an mTOR-mediated link between purinosomes and mitochondria, and a general means by which mTOR regulates nucleotide metabolism by spatiotemporal control over protein association.

Spatial control of cellular enzymes

Biochemistry Purine is a building block of DNA and also a component of ATP that is used as an energy source in the cell. Enzymes involved in purine biosynthesis organize into dynamic bodies called purinosomes. French et al. found that purinosomes colocalize with mitochondria, organelles that

HIV-1 Reverse Transcriptase-Based Assay to Determine Cellular dNTP Concentrations.

Deoxynucleoside triphosphates (dNTPs) are the building blocks of DNA and their biosynthesis is tightly regulated in the cell. HPLC-MS and enzyme-based methods are currently employed to determine dNTP concentrations from cellular extracts. Here, we describe a highly efficient, HIV-1 reverse transcriptase (RT)-based assay to quantitate dNTP concentrations. The assay is based on the ability of HIV-1 RT to function at very low dNTP concentrations, thus providing for the high sensitivity of detection.

A rapidly photo-activatable light-up fluorescent nucleoside and its application in DNA base variation sensing.

A new DNA building block (d(Tet)U) bearing a tetrazole and allyloxy group at N-phenyl ring linked through an aminopropynyl linker to the 5-position of 2'-deoxyuridine was synthesized. The modified DNA can be lit up via a photoinduced intramolecular tetrazole-alkene cycloaddition reaction, but quenched when the fully-matched double strand is formed. This conspicuous difference in fluorescence could open a door for DNA single nucleotide polymorphism (SNP) typing.

Surface Stiffness--a Parameter for Sensing the Chirality of Saccharides.

Surface stiffness is considered a key parameter for designing high-performance implantable materials and artificial extracellular matrices because of its substantial effects on cell behavior. How to transform biomolecule recognition events, particularly chiral recognition, into stiffness change on material surfaces is biologically essential but very challenging for chemists. Here, we report a chirality-triggered stiffness transition on a smart polymer film, which consists of flexible polyethylenimine (PEI) main chains grafted with dipeptide units capable of discriminating chiral monosaccharides. The polymer film became substantially softer after interacting with L-ribose and became more rigid after interacting with D-ribose (the basic building block of DNA and RNA). This chiral effect provides a new method for determining the enantiomeric purity of an L/D-ribose mixture and facilitates the chiral separation of deoxyribose racemates as well as the separation of diverse mono-, di-, and oligosaccharides. These are three puzzle problems in carbohydrate chemistry. Furthermore, taking advantage of the significant differences in the surface stiffness, the proliferation of fibroblast cells on the polymeric surfaces can also be regulated by chiral biomolecules.

Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis.

Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication.

Reactions of Azine Anions with Nitrogen and Oxygen Atoms: Implications for Titan's Upper Atmosphere and Interstellar Chemistry.

Azines are important in many extraterrestrial environments, from the atmosphere of Titan to the interstellar medium. They have been implicated as possible carriers of the diffuse interstellar bands in astronomy, indicating their persistence in interstellar space. Most importantly, they constitute the basic building blocks of DNA and RNA, so their chemical reactivity in these environments has significant astrobiological implications. In addition, N and O atoms are widely observed in the ISM and in the ionospheres of planets and moons. However, the chemical reactions of molecular anions with abundant interstellar and atmospheric atomic species are largely unexplored. In this paper, gas-phase reactions of deprotonated anions of benzene, pyridine, pyridazine, pyrimidine, pyrazine, and s-triazine with N and O atoms are studied both experimentally and computationally. In all cases, the major reaction channel is associative electron detachment; these reactions are particularly important since they control the balance between negative ions and free electron densities. The reactions of the azine anions with N atoms exhibit larger rate constants than reactions of corresponding chain anions. The reactions of azine anions with O atoms are even more rapid, with complex product patterns for different reactants. The mechanisms are studied theoretically by employing density functional theory; spin conversion is found to be important in determining some product distributions. The rich gas-phase chemistry observed in this work provides a better understanding of ion-atom reactions and their contributions to ionospheric chemistry as well as the chemical processing that occurs in the boundary layers between diffuse and dense interstellar clouds.

SEEDING THE PREGENETIC EARTH: METEORITIC ABUNDANCES OF NUCLEOBASES AND POTENTIAL REACTION PATHWAYS

Carbonaceous chondrites are a class of meteorite known for having a high content of water and organics. In this study, abundances of the nucleobases, i.e., the building blocks of RNA and DNA, found in carbonaceous chondrites are collated from a variety of published data and compared across various meteorite classes. An extensive review of abiotic chemical reactions producing nucleobases is then performed. These reactions are then reduced to a list of 15 individual reaction pathways that could potentially occur within meteorite parent bodies. The nucleobases guanine, adenine and uracil are found in carbonaceous chondrites in the amounts of 1$-$500 ppb. It is currently unknown which reaction is responsible for their synthesis within the meteorite parent bodies. One class of carbonaceous meteorites dominate the abundances of both amino acids and nucleobases$-$the so-called CM2 (e.g. Murchison meteorite). CR2 meteorites (e.g. Graves Nunataks) also dominate the abundances of amino acids, but are the least abundant in nucleobases. The abundances of total nucleobases in these two classes are $330 \pm250$ ppb and $16 \pm13$ ppb respectively. Guanine most often has the greatest abundances in carbonaceous chondrites with respect to the other nucleobases, but is 1$-$2 orders of magnitude less abundant in CM2 meteorites than glycine (the most abundant amino acid). Our survey of the reaction mechanisms for nucleobase formation suggests that Fischer-Tropsch synthesis (i.e. CO, H$_2$ and NH$_3$ gases reacting in the presence of a catalyst such as alumina or silica) is the most likely for conditions that characterize early states of planetesimals.

Nitrogen limitation as a driver of genome size evolution in a group of karst plants.

Genome size is of fundamental biological importance with significance in predicting structural and functional attributes of organisms. Although abundant evidence has shown that the genome size can be largely explained by differential proliferation and removal of non-coding DNA of the genome, the evolutionary and ecological basis of genome size variation remains poorly understood. Nitrogen (N) and phosphorus (P) are essential elements of DNA and protein building blocks, yet often subject to environmental limitation in natural ecosystems. Using phylogenetic comparative methods, we test this hypothesis by determining whether leaf N and P availability affects genome sizes in 99 species of Primulina (Gesneriaceae), a group of soil specialists adapted to limestone karst environment in south China. We find that genome sizes in Primulina are strongly positively correlated with plant N content, but the correlation with plant P content is not significant when phylogeny history was taken into account. This study shows for the first time that N limitation might have been a plausible driver of genome size variation in a group of plants. We propose that competition for nitrogen nutrient between DNA synthesis and cellular functions is a possible mechanism for genome size evolution in Primulina under N-limitation.

Sequential growth of long DNA strands with user-defined patterns for nanostructures and scaffolds.

DNA strands of well-defined sequence are valuable in synthetic biology and nanostructure assembly. Drawing inspiration from solid-phase synthesis, here we describe a DNA assembly method that uses time, or order of addition, as a parameter to define structural complexity. DNA building blocks are sequentially added with in-situ ligation, then enzymatic enrichment and isolation. This yields a monodisperse, single-stranded long product (for example, 1,000 bases) with user-defined length and sequence pattern. The building blocks can be repeated with different order of addition, giving different DNA patterns. We organize DNA nanostructures and quantum dots using these backbones. Generally, only a small portion of a DNA structure needs to be addressable, while the rest is purely structural. Scaffolds with specifically placed unique sites in a repeating motif greatly minimize the number of components used, while maintaining addressability. This combination of symmetry and site-specific asymmetry within a DNA strand is easily accomplished with our method.

Alternative Thymidylate Synthase: New Intermediates Suggest New Flavin Mechanism

Flavin-dependent, thymidylate synthase (FDTS, ThyX) catalyzes the last crucial step in the de novo biosynthesis of thymidylate, a DNA building block. Because FDTS is absent in humans while present in several human pathogens, its inhibition is of interest from pharmaceutical standpoint. Unlike classical thymidylate synthase (TSase, ThyA), FDTS bypasses the need for an active-site nucleophile.1 FDTS still has to activate the substrate for the reaction; however, the nucleophile is not an enzymatic residue as in TSase.2 Further understanding of the chemical mechanism of FDTS would greatly facilitate rational design of mechanism-based inhibitors as leads for new class of antibiotic drugs. We will present the trapping of several intermediates3 that shad new light on possible mechanism for this enzyme. By using acid and base as a quenchers in a rapid-quench experiment, we find that the first intermediate yields different trapped compounds in different quesnchers. The second intermediate, on the other hand, forms dTMP product upon base quenching, which is surprising given that the flavin cofactor is still reduced at this stage (according to flavin's UV-vis absorbance spectrum). The recent identification of the base-trapped intermediate and a crystal structure suggest a new path for the catalyzed methylene transfer from the co-factor (methylene tetrahydrofolate) and the substrate dUMP. These new findings may point to a methylene transfer from its folate-donor to the target nucleotide through N5 of the FAD cofactor, in accordance with findings from X-ray crystallography.4 Funding NIH RO1 GM110775

Conformation and orientation dependence in ion-induced collisions with DNA and RNA building blocks

Action of radiations on biological tissues is of major concern in cancer therapy development. Understanding the mechanisms involved at the molecular level in such reactions may be of crucial interest. In particular ion-induced ionization processes appear at the early stage of damage and a detailed analysis has been performed on the charge transfer dynamics of carbon ions with the different DNA and RNA building blocks in order to analyze their respective behavior in ion-induced collisions. We have considered the pyrimidine nucleobases uracil and thymine and the 5-halouracil molecules corresponding to the same skeleton, as well as the sugar moiety 2-deoxy-D-ribose. The calculations have been performed by means of ab initio quantum chemistry molecular methods followed by a semi-classical collision treatment in a wide collision energy range. Considerations of the structure of the biological target as well as analysis of the anisotropy of the process have been performed. The comparison with proton collisions has been developed with regard to previous results. Qualitative trends of interest for DNA building blocks damage may be pointed out.

Options for the treatment of HIV: A review

C has estimated that approximately 1.2 million people in the United States were living with HIV infection at the end of 2011 (the most recent year for which data are available).1 Of those 1.2 million people, around 1 in 7—close to 170,000—were unaware of their infection status.1 For pharmacists who do not regularly dispense medications for the treatment of HIV, understanding treatment complexities for these patients can be intimidating.