DNA Binding Kinetics of Large Antiviral Hairpin Polyamides

While vaccines exist for the some of the most problematic strains of human papillomavirus (HPV), a double stranded DNA virus, there is currently no cure. HPV remains one of the most commonly sexually transmitted infections and is responsible for virtually all cervical cancers and genital warts. Natural products Distamycin A and netropsin have inspired the hairpin Nmethylpyrrole (Py)/N-methylimidazole (Im) polyamides (PAs) studied here. The larger hairpin PAs, designed to bind to sites of 10 or more DNA bp, have been shown to be effective antivirals against oncogenic HPV strains 16, 18, and 31, while smaller hairpin PAs are not. Despite significant differences in potencies among the antiviral PAs tested, the PAs bind to DNA with similar binding affinity (Kd). Ample evidence has shown dissociation rate constants (koff) may be a better indicator of drug efficacy than Kd. While Kd is a function of association rate constant (kon) and koff, respectively (Kd = koff/kon), few studies have focused on the DNA binding kinetics of large hairpin PAs. We are using fluorescence and CD spectroscopy to characterize Kds, obtain DNA binding kinetic rate constants, and determine binding stoichiometries as a function of PA size. K= remains tight (low nM) for all PAs tested (6-20-rings) with our fluorescence assay, which is consistent with what is seen with other methods. The large PAs are characterized by slow DNA dissociation rates with half-lives ranging from 20-30 min, and dissociation slows as the size of PAs increases. Slow dissociation rates are likely the source of the difference in antiviral behavior. Association time courses for a 14-ring PA indicate that \u3e1 equivalent of PA is binding to the DNA. Further supporting this claim, fluorescence and CD spectroscopic experiments indicate that saturation of the DNA does not occur until 2 or more equivalents of PA are added. The slow dissociation of multiple equivalents of PA bound to DNA may cause a large and prolonged disruption in DNA conformation, which in turn elicits or alters the DNA damage response, which is an integral part in the antiviral mechanism of large antiviral hairpin PAs and of the lifecycle of HPV

Polyamides containing amino butyric acid-based building blocks

The present invention relates to polyamide compositions and therapies for treating cells infected with papilloma virus. For the most up-to-date information about these patents, including the availability of Certificates of Correction, be sure to check the United States Patent and Trademark Office\u27s free, publicly accessible database: Patent Public Search https://ppubs.uspto.gov/pubwebapp/static/pages/landing.htmlhttps://irl.umsl.edu/patents/1030/thumbnail.jp

Methods for treating papilloma virus infection

For the most up-to-date information about these patents, including the availability of Certificates of Correction, be sure to check the United States Patent and Trademark Office\u27s free, publicly accessible database: Patent Public Search https://ppubs.uspto.gov/pubwebapp/static/pages/landing.htmlhttps://irl.umsl.edu/patents/1024/thumbnail.jp

Polyamides for treating human papilloma virus

The present invention relates to polyamide compositions and therapies for treating cells infected with human papilloma virus (HPV). For the most up-to-date information about these patents, including the availability of Certificates of Correction, be sure to check the United States Patent and Trademark Office\u27s free, publicly accessible database: Patent Public Search https://ppubs.uspto.gov/pubwebapp/static/pages/landing.htmlhttps://irl.umsl.edu/patents/1031/thumbnail.jp

Molecular Dynamics Simulation Studies of DNA and Proteins: Force Field Parameter Development of Small Ligands and Convergence Analysis for Simulations of Biomolecules

In the first part of this dissertation, CHARMM force field parameters for DNA minor groove-binding polyamides were developed. The parameterization involved the subdivision of the polyamides into model compounds, which were calibrated against MP2/6-31G(d) data. To test the new parameters, fourteen 10 ns molecular dynamics crystal simulations have been carried out on a DNA/polyamide complex at low (113K) and high (300K) temperatures. Of the 18 helical parameters examined, only one (stagger) is found to be statistically significant from the crystal structure with a t-test at the 95% confidence level. For the high temperature, stagger is non-significant at the 97% confidence level, which underscores the importance of running multiple trajectories. It is observed that when the simulations are run at 300K, the DNA fragment begins to distort; however, better sampling is achieved. Competition between water and polyamides for hydrogen bonding to DNA is found to explain weak or unpredictable binding. In the second part, force field parameters for retinoids were developed. The retinoids were divided into model compounds and calibrated against MP2/6-31G(d) data. To test the parameters, five molecular dynamics crystal simulations of reported x-ray structures of protein/retinoid complexes were performed. The structural and geometric analysis of these simulations compares well to experiment, and some dynamics that could be important to ligand binding were discovered. The new parameters can now be used in simulations of retinoid-binding proteins to better understand these systems and in drug design to make new retinoids with therapeutic and anticancer potential. The last part explores the convergence of structural parameters in biomolecular systems. A simple statistical test was applied to the different parameters from a few long and many short simulations to observe which strategy is best. For the protein, both the long and short simulations gave similar results with respect to convergence. For the DNA, it was found that fraying effects penetrate four base pairs in from the ends of the helix. Structural parameters converge more quickly for the middle four bases than for all bases, and the long simulations yielded better results with respect to convergence than the short simulations

Hope College Abstracts: 11th Annual Celebration of Undergraduate Research and Creative Performance

The abstracts...are representative of student-faculty collaborative research and creative work that takes place throughout the year at Hope

REPSA Directed Assessment of Native Cleavage Resistance of DNA to Type IIS Restriction Endonucleases and Modification of REPSA for High Temperature Application

We have modified the combinatorial selection method Restriction Endonuclease Protection and Selection Assay (REPSA) to work in high temperature conditions for the discovery of new DNA-binding proteins in thermophiles (HT-REPSA). We utilized Thermus thermophilus (HB-8/ATCC 27634/DSM 579) as a test organism due to its amenable nature in a laboratory setting and current status as a model thermophilic organism. We used a TetR Family (TFR) transcription factor SbtR as the model protein for optimization of HT-REPSA protocols, as data had previously been obtained regarding SbtR physical characteristics and DNA-binding properties. REPSA was conducted until a cleavage resistant species arose after 7 rounds. Massively parallel sequencing of the selected DNAs and bioinformatics analysis yielded a consensus binding sequence of 5\u27-GA(t/c)TGACC(c/a)GC(t/g)GGTCA(g/a)TC, a 20base pair palindromic site comparable to that described in the literature. Taken together, our data provide a proof-of-concept that HT-REPSA can be successfully used to identify the preferred DNA-binding sequences of transcription factors from extreme thermophilic organisms

Shielding and mediating of hydrogen bonding in amide-based (macro)molecules

Polymers are long chain molecules comprising continuously repeating building blocks, monomers, which are chemically linked via covalent bonds, for example the C-C bond in polyethylene. A distinction can be made in biopolymers that are made in nature and synthetic polymers that are produced by the chemical industry (plastics). Properties of polymeric materials are not only determined by the primary chemical structure, i.e. the chemical composition of the polymer chain, but also by the secondary interactions between the chains (intermolecular interactions) and the conformation (shape). Especially in biopolymers, a delicate balance between the primary chemical structure, i.e. the chain composition, and intra- and intermolecular interactions is encountered. A well known example is the double helix in DNA, which carries the essence of life. Another example is peptides, or proteins, where unique conformations are dependent on a balance between the sequence of monomer units, here amino acids, and secondary interactions (e.g. hydrogen bonding) between monomers in a single molecule, the formation of the known a- helix and ß-sheet structures, and/or between molecules. Synthetic polymers are in comparison to biopolymers chemically less sophisticated, rendering higher thermal stability. Hence, synthetic polymers can be directly processed via melt routes into end products, for example by means of injection molding or extrusion, while biopolymers such as cellulose (wood) have to be chipped. The conformation and secondary interactions between molecules are essential in synthetic polymers as well. An extreme example in this respect is the simplest polymer on earth: polyethylene (PE). Taking polyethylene as precursor, the industry produces flexible films and containers on one hand, and superstrong fibers with a specific strength and stiffness larger than steel on the other. In these fibers all polymer molecules exist in extended chain conformation perfectly aligned in the fiber direction. Between the apolar PE molecules only relatively weak van der Waals forces reside, but with sufficient length of the molecules the sum of the weak secondary interactions between the molecules induces sufficient frictional forces between the chains that the stress is transferred to the covalent bonds in the main chain upon deformation (in the fiber direction), resulting in high strength and stiffness. These superstrong polyethylene fibers can be considered as 1-dimensional diamond at fast deformation rates, whereas at low deformation rates, i.e. long time-scales, creep occurs. Another disadvantage of these PE fibers is the relatively low melting temperature, approximately 150°C. More ideal would be the use of polyamides (nylons) as precursor for superstrong fibers since polyamides prevent creep by hydrogen bonding and possess high melting temperatures. Chemically polyamides are similar to proteins where the monomers are connected by amide moieties. Polymer chemists often describe proteins as decorated nylons (nylon 2). In the past a lot of industrial research effort has been addressed to the development of superstrong polyamide fibers in a similar way as PE, i.e. drawing and aligning the chains in the fiber direction, unfortunately without success. In processing polyamides, either from the melt or from solution, cooling induces crystallization into chain folded crystals that are comparable with stacks of ß-sheets in proteins. Because of relatively strong interchain hydrogen bonding the chain folded crystals cannot be unfolded like in the case of polyethylene. The aim of the thesis is to shield hydrogen bonding in polyamides temporarily during processing and drawing and to restore the hydrogen bonding once the chains are ideally aligned and extended. Based on the dissolution of polyamides in the superheated state of water (PhD thesis Esther Vinken, TU/e 2008) and inspired by natural silk spinning, where in the glands of spiders and silk worms hydrogen bonded moieties of the proteins are shielded and mediated by water molecules, salts (ions) and pH, a new reversible shielding route in polyamide processing is introduced. Since the amorphous phase in polyamides imposes limitations in investigating the role of water molecules on crystalline hydrogen bonding in polyamides after crystallization from the superheated state of water, low molar mass model compounds, expected to represent the crystalline domains in aliphatic polyamides, have been studied in the dissolution in, and crystallization from the superheated state of water (chapters 2, 3 and 4). The model molecules are bisamide-diols, possessing two central amide motifs (head-tohead) and two hydroxylic end groups. The aliphatic segment length, which separates the polar moieties, can be varied in analogy to polyamides. Hydrogen bonding in polyamides resides in the structural amide planes, which stack to form chain folded crystals. A bisamide-diol with a short aliphatic segment between the amide motifs combined with two longer identical segments between the amide and hydroxyl moieties crystallizes in a stack of crystalline planes in which the molecules are held together by amide-amide hydrogen bonding. In case of a rather equal segment length between all polar groups in the bisamide-diol, amide-hydroxyl hydrogen bonding occurs between the structural amide planes. Hence, the role as a model compound is questionable in such a scenario. Nevertheless, the thermodynamic, structural and conformational behavior is, identical to polyamides, dependent on a balance between thermal motion and hydrogen bonding efficiency. Both bisamide-diols are soluble in the superheated state of water. During crystallization upon cooling the interaction of water molecules with the amide motifs erases the conformational limitations of the intrinsically rigid amide moieties. The extra degree of freedom during crystallization entails ideal crystalline hydrogen bonding, stabilizing the crystalline structures. Moreover, water molecules can be trapped within the crystal lattice during crystallization. In the second part of the dissertation, water molecules are assisted by a series of Hofmeister ions in shielding and mediating of hydrogen bonding in polyamides. The Hofmeister series is a classification based on the hydrating nature of ions, known as kosmotropic and promoting the organization of water molecules, or non-hydrating character of ions, referred to as chaotropic and disordering the water structures. Close to the Brill transition temperature, a reversible crystal transformation that arises due to variations in aliphatic molecular motion and hydrogen bonding efficiencies, polyamides can be dissolved in the superheated state of water. With increasing ionic strength large non-hydrating ions of halogenic origin, such as bromide and iodide, perturb the hydrogen bonding network between water molecules. Since the diffusivity of water molecules and its solutes increases, water molecules and small strongly hydrating cations penetrate the polyamide crystal at lower temperatures, perturbing the amide-amide hydrogen bonding in the crystal. Next to the suppression of the dissolution temperature, the crystallization temperature upon cooling decreases as well. To minimize the nonpolar surface area, hydrophobic hydration entails secretion of the anions to the hydrophobic methylene segments at high ionic strength. With the interaction of the cations, preferably lithium, a charge distribution along the polyamide chains is formed that suppresses crystallization even at room temperature. Extensional deformation of the aqueous polyamide solution in excess of water results in the migration of ions, restitution of intermolecular hydrogen bonding and orientation. However, although the aqueous solutions can be deformed into drawable filaments, the strength upon crystallization is lost due to the absence of chain overlap, meaning that stress transfer between the chains is insufficient. To promote chain overlap a processing (extrusion) route for concentrated polyamide LiI solutions is explored in chapter 6. Here, the hydrogen bonding is temporarily shielded by ions to prevent the formation of chain folded crystals during processing. Strain induced crystallization upon drawing restores amide-amide hydrogen bonding, high orientation factors and lattice perfection. Though these aspects are essential in realizing high strength and high modulus materials, the crystallinity and the melting temperature are considerably suppressed by incomplete removal of ions. Time-resolved wide angle X-ray experiments reveal that the migration of ions is primarily time-dependent at temperatures above the glass transition temperature. Efficient migration of ions in superheated water at 150°C results in high crystallinities and consequential high melting temperatures, preserving the high orientation and crystal perfection. However, experimental verification of the ultimate goal: the development of high strength and stiff polyamide fibers, could not be realized due to the intrinsic problem of removing all ions effectively that requires optimization in terms of fiber diameter and spin/drawing parameters. Ideal experimental conditions are of technological origin and require optimization in an industrial environment. The author hopes that the results in this dissertation will contribute to a new technology resulting to a new generation of (super)strong polyamide fibers

Multistep reactions based on thiolactones for the synthesis of functionalized polymers

The aim of this research project was to develop a method for the synthesis of advanced polymeric structures without the use of timeconsuming protecting group strategies. The proposed method is based on the reaction between thiols and unsaturated carbon chains, denoted as thiol-ene or thiol-yne chemistry. Because of the disadvantages of working with thiols (i.e. unpleasant smell, limited commercial availability and instability due to oxidation reactions), there has been a continuous interest in the development of new ways to protect thiols, e.g. as disulfide, thiocarbonylthio-group or methanesulfonate. However, most of these methods require a protecting and a deprotecting step, which is unfavorable in terms of atom-efficiency and overall yield. The strategy herein presented, is based on the use of a thiolactone as a precursor for a thiol, allowing for the direct introduction of a thiol, starting from stable amino compounds without the need for a subsequent deprotection step. Using thiolactones in polymer synthesis has a dual advantage: On the one hand, it offers a chemoselective, atom-efficient way of generating thiols, while at the same time it is possible to introduce functionality via the amine compound. This one-pot amine-thiol-ene conjugation reaction was used for the synthesis and modification of diverse polymeric systems