Enzymová syntéza DNA modifikované v malém žlábku

In the first part of the thesis, a series of six modified 2'-deoxyadenosine triphosphates, bearing small functional groups (chloro, amino, methyl, vinyl, ethynyl and phenyl) at position 2 of adenine, was designed and synthesised. They were then tested as substrates for DNA polymerases in enzymatic synthesis of minor-groove modified DNA. The 2-phenyl modified dATP was the only triphosphate unable to be incorporated, meaning that the phenyl group is already too big for minor-groove incorporations. All of the other tested nucleotides were good substrates for tested DNA polymerases [KOD XL, Vent(exo-) and Bst LF] affording minor- groove modified DNA bearing one or four modifications. The vinyl- and ethynyl-modified DNAs were then used for post-synthetic modification of DNA minor groove with fluorescent labels utilising click reactions. Ethynyl group reacted in copper-catalysed alkyne-azide cycloaddition (CuAAC), whereas the vinyl group participated in thiol-ene reaction. This procedure allowed for the attachment of big functional groups otherwise unable to be installed into the DNA minor groove using direct enzymatic incorporation. The second part of the thesis was devoted to the study of 2-alkylamino-2'- deoxyadenosine triphosphates and their use in enzymatic synthesis of base-modified ONs and DNA....Souhrn V první části práce byla navržena a syntetizována řada šesti modifikovaných 2'- deoxyadenosintrifosfátů, nesoucí malé funkční skupiny (chlor, amino, methyl, vinyl, ethynyl a fenyl) na adeninu v poloze 2. Připravené nukleotidy byly testovány jako substráty pro DNA polymerázy v enzymatické syntéze DNA modifikované v malém žlábku. 2-Fenyl modifikovaný dATP byl jediným trifosfátem, který nebyl inkorporovaný, což znamená, že fenylová skupina je již příliš velká pro inkorporaci do malého žlábku. Všechny ostatní testované nukleotidy byly dobrými substráty pro testované DNA polymerázy [KOD XL, Vent(exo-) a Bst LF], což poskytlo modifikovanou DNA s malým žlábkem nesoucím jednu nebo čtyři modifikace. Modifikované DNA s vinylovou nebo ethynylovou skupinou byly poté použity pro modifikaci malého žlábku DNA fluorescenčními značkami za použití click reakcí. Ethynylová skupina reagovala v mědí katalyzované cykloadici alkynu s azidem (CuAAC), zatímco vinylová skupina se účastnila thiol- en-ové reakce. Tento postup umožnil připojení velkých funkčních skupin, které by jinak nemohly být instalovány do malého žlábku DNA pomocí přímé enzymatické inkorporace. Druhá část práce byla věnována studiu 2-alkylamino-2'-deoxyadenosin trifosfátů a jejich použití v enzymatické syntéze modifikovaných oligonukleotidů a DNA. N2 -alkyl...Department of Organic ChemistryKatedra organické chemiePřírodovědecká fakultaFaculty of Scienc

Enzymatic synthesis of DNA modified in the minor groove

In the first part of the thesis, a series of six modified 2'-deoxyadenosine triphosphates, bearing small functional groups (chloro, amino, methyl, vinyl, ethynyl and phenyl) at position 2 of adenine, was designed and synthesised. They were then tested as substrates for DNA polymerases in enzymatic synthesis of minor-groove modified DNA. The 2-phenyl modified dATP was the only triphosphate unable to be incorporated, meaning that the phenyl group is already too big for minor-groove incorporations. All of the other tested nucleotides were good substrates for tested DNA polymerases [KOD XL, Vent(exo-) and Bst LF] affording minor- groove modified DNA bearing one or four modifications. The vinyl- and ethynyl-modified DNAs were then used for post-synthetic modification of DNA minor groove with fluorescent labels utilising click reactions. Ethynyl group reacted in copper-catalysed alkyne-azide cycloaddition (CuAAC), whereas the vinyl group participated in thiol-ene reaction. This procedure allowed for the attachment of big functional groups otherwise unable to be installed into the DNA minor groove using direct enzymatic incorporation. The second part of the thesis was devoted to the study of 2-alkylamino-2'- deoxyadenosine triphosphates and their use in enzymatic synthesis of base-modified ONs and DNA...

Investigation into mechanical, surface and adhesive properties of date palm wood-polyolefin micro composites

Wood-plastic composites are composite materials made of wood fibre or other lignocellulosic materials and thermoplastic(s). Date palms are one of the potential replacements of insufficient timber sources in the Middle East and the Horn of Africa countries. Low density polyethylene (LDPE) was blended with date palm wood (DPW) (Phoenix dactylifera) powder to prepare composites with the concentrations of filler ranging from 10 to 70 wt. %. The Young´s modulus of the composites significantly increased with an increase in the filler content in the entire concentration range. The maximum value of 1933 MPa for the composite filled with 70 wt.% of the filler is approximately 13 times higher than that for the neat LDPE. The incorporation of DPW into the LDPE matrix led to a significant increase in the polarity of composites and to an increase in their adhesion to polar substrates.This contribution was supported by Ministry of Education of the Slovak Republic and Slovak Academy of Sciences, project VEGA, Grant No. 1/0570/17. This research was supported by the Slovak Research and Development Agency under the contracts No. APVV-16-0177, APVV-17-0583 and APVV-18-0378.Scopu

Optimization of raw hide curing using two-component counter-current diffusion model

Ošetření koncentrovaným roztokem solanky (chlorid sodný) je běžná metoda konzervace surových kůží. Sodné a chloridové ionty difundují ve směru do vnitřního objemu kůže, což iniciuje protiproudý transport vody směrem k povrchu. Výsledkem dvousložkové protiproudé difúze je rostoucí koncentrace soli a klesající obsah vody ve vnitřním objemu solené kůže. Pro optimalizaci konzervačního procesu je nezbytné zjistit časovou závislost koncentrace solanky směrem od povrchu do vnitřního objemu, to znamená nestacionární koncentrační pole ve vnitřním objemu konzervované kůže. Pro tyto účely byl navržen matematicko-fyzikální model difúze NaCl v kůži. Bylo provedeno numerické řešení tohoto modelu ? toto řešení zahrnuje koncentrační pole chloridu sodného a vody během konzervačního procesu. Byly experimentálně stanoveny příslušné efektivní difúzní koeficienty a jejich závislost na koncentraci soli. Výsledkem matematické simulace a matematického zhodnocení experimentálních dat byl odhad optimálního času pro solení v daných okrajových podmínkách, a dostatečné koncentrace soli ve vnitřním objemu solené kůže.Curing with concentrated brine solution (sodium chloride) is a common method for raw hide preservation. Sodium and chloride ions diffuse towards the raw hide inner volume, which initiates the counter-current transport of water from the raw hide inner volume. The result of the two-component counter-current diffusion is increasing salt content and decreasing water content in the raw hide inner volume. For optimization of the curing process it is necessary to determine the time dependence of the brine concentration from the hide surface towards its center, it means the non-stationary concentration field in the inner volume of the hide. For this reason we have proposed a mathematical-physical model for NaCl diffusion in the hide. We have also carried out the numerical solution of the modelthe solution comprises the concentration fields of sodium chloride and water during the curing process. The respective effective diffusion coefficients and their dependence on brine concentration have been determined experimentally. The result of the mathematical simulation and mathematical evaluation of the experimental data was estimation of the optimal time for raw hide curing at the given boundary conditions, and a sufficient brine concentration in the inner volume of the hide

Cold Plasma-An Efficient Tool for Nanotechnological Modification of Polyester-Based Polymers

Polyester surface was pretreated by cold plasma to improve its adhesive properties. The changes in the chemical structure of the pretreated polymer were analyzed by attenuated total reflectance Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. The changes in the polyester surface morphology were studied using scanning electron microscopy and atomic force microscopy. The surface energy, and its polar contribution, as well as the peel strengths of the adhesive joints of polyester, modified by cold plasma to polyacrylate, increased significantly. The efficiency of the material modification depends on a type of discharge, the used gases, the power of plasma source, as well as on the time of modification treatment. Aging of the polymer modified by plasma was also investigated. A correlation between adhesion of polyester modified by cold plasma to polyacrylate and its surface energy was found.Scopu

Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases

Abstract Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases – SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics

Investigation of selected anti-bacterially modified Micro/Nano-polymers using cold plasma

Medical-grade polymers, i.e., polyvinylchloride and low-density polyethylene (LDPE), were modified by antibacterial compounds through a multistep physicochemical approach. The polymer initial surface activation was performed foremost via diffuse coplanar surface barrier discharge (DCSBD) plasma in air at ambient temperature and atmospheric pressure. Then, radical graft copolymerization of acrylic acid (AA) through grafting-from pathway was directed to render a well-defined brush of high density, and finally the selected antibacterial compounds were immobilized onto the functionalized polymer surfaces. This modification led to inhibition of Escherichia coli and Staphylococcus aureus bacterial strains adhesion to the polymer surface.Scopu

Antibacterial polyvinylchloride pre-treated by barrier plasma

A multistep physicochemical approach making use of plasma technology combined with wet chemistry has fueled considerable interest in delivery of surface-active antiadherence materials. In the first step of the approach, concerning an inherent lack of befitting functional groups on pristine substrate, plasma treatment at low temperature and atmospheric pressure has been substantiated to be productive in yielding reactive entities on the surface [1, 5]. The highlights the functionality of the adopted multistep physicochemical approach to bind polysaccharide species onto the medical-grade PVC surface. DCSBD plasma is capable of raising roughness, surface free energy, and introducing oxygen-containing functionalities anchored onto the surface. A structured poly(acrylic acid) brush of high graft density is synthesized using surface-initiated approach to further improve hydrophilicity and develop a stable brush-like assembly to yield a platform for biomolecular binding. In vitro bacterial adhesion and biofilm formation assays indicate incapability of single chitosan layer in hindering the adhesion of Staphylococcus aureus bacterial strain. Chitosan could retard Escherichia coli adhesion and plasma treated and graft copolymerized samples are found effective to diminish the adherence degree of Escherichia Coli. © 2014 by Nova Science Publishers, Inc. All rights reserved

Nucleotides bearing aminophenyl- or aminonaphthyl-3-methoxychromone solvatochromic fluorophores for the enzymatic construction of DNA probes for the detection of protein–DNA binding

Two new 2'-deoxyribonucleoside triphosphates bearing solvatochromic fluorophores were prepared and used for enzymatic synthesis of DNA probes that light-up and change colour upon interactions with proteins.We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores showed solvatochromic fluorescence with strong fluorescence at 433-457 nm in non-polar solvents and very weak fluorescence at 567 nm in alcohols. The nucleosides and nucleotides also showed only negligible fluorescence in alcohols or water. The triphosphates were substrates for DNA polymerase in the enzymatic synthesis of modified DNA probes that showed only very weak fluorescence in aqueous buffer but a significant light-up and blue shift were observed when they interacted with proteins (histone H3.1 or p53 for double-stranded DNA probes or single-strand binding protein for single-stranded oligonucleotide probes). Hence, nucleotides have good potential in the construction of DNA sensors for studying protein-DNA interactions. The modified dNTPs were also transported into cells using a cyclodextrin-based transporter but they were not incorporated into the genomic DNA