Scission Fragments Research Articles

Quantification and Statistical Analysis of Errors Related to the Approximate Description of Active Site Models in Metal‐Exchanged Zeolites

AbstractMetal‐exchanged zeolites are complex catalytic systems with great potential for applications in hydrocarbon conversion processes. The numerous combinations of zeotypes and metal‐based Lewis acid motifs create a vast material space that can only be effectively navigated with robust structure‐function relationships. Exhaustive modeling of extraframework metal sites in all crystallographically distinct framework positions is not practical; thus, we quantified the uncertainties arising from the use of simplified active site representations using the C−H bond activation in methane as a probe reaction. Density functional theory calculations and statistical analysis of associated error ensembles suggest that the Lewis acid identity primarily determines the reactivity of the zeolite towards methane activation, with other factors such as zeolite model characteristics deemed secondary. The error ensemble distributions were used to predict the relative probability of creating active C−H bond scission fragments in metal‐exchanged zeolites, and thus can be used to efficiently screen various metal‐exchange candidates in their efficacy towards hydrocarbon conversion.

Systematic Analysis of Fission Fragment Mass Distribution and TKE for Actinides by Langevin Equation

Dynamical description of fission by Langevin equations is carried out for several actinides to see systematical trends in fission fragment mass and kinetic energy distributions. The transport coefficients are calculated by both macroscopic and microscopic theories. The light and heavy scission fragment mass average <AL> and <AH> are compared with the original Flynn’s <AL> and <AH>. We also find a reasonable agreement of our calculated <AL> and <AH> with the corresponding averages from evaluated data of JENDL’s 14 MeV incident neutron fission product yield. We compare the calculated total kinetic energy of the fission fragment with Viola’s systematics. Good agreements are also seen for the actinides under study in comparison with total kinetic energy from JENDL. Effects of the microscopic transport coefficients on these systematical trends are discussed.

Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination

ABSTRACTBy combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids.

Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter

DNA manipulations using a completely chemistry-based DNA cutter (ARCUT) have been reviewed. This cutter, recently developed by the authors, is composed of Ce(IV)/EDTA complex and two strands of pseudo-complementary peptide nucleic acid. The site-selective scission proceeds via hydrolysis of targeted phosphodiester linkages, so that the resultant scission fragments can be easily ligated with other fragments by using DNA ligase. Importantly, scission-site and site-specificity of the cutter are freely tuned in terms of the Watson–Crick rule. Thus, when one should like to manipulate DNA according to the need, he or she does not have to think about (1) whether appropriate “restriction enzyme sites” exist near the manipulation site and (2) whether the site-specificity of the restriction enzymes, if any, are sufficient to cut only the aimed position without chopping the DNA at non-targeted sites. Even the human genome can be manipulated, since ARCUT can cut the genome at only one predetermined site. Furthermore, the cutter is useful to promote homologous recombination in human cells, converting a site to desired sequence. The ARCUT-based DNA manipulation should be promising for versatile applications.

Artificial site-selective DNA cutters to manipulate single-stranded DNA

Recent progress regarding the development of artificial site-selective DNA cutters by chemical approaches are reviewed herein, with a special focus on site-selective cutters for single-stranded DNA. We employ a Ce(IV)/EDTA complex to serve as the catalyst, because it efficiently and selectively cuts single-stranded DNA. Using two complementary oligonucleotide additives, a gap structure is formed at the target site in the single-stranded DNA substrate. Owing to the substrate specificity of Ce(IV)/EDTA, the gap site is preferentially hydrolyzed, resulting in a site-selective DNA scission. The scission site is easily determined using the Watson–Crick base-pairing rule; thus, both the sequence and scission specificity can be tuned according to demand. The site-selective scission is greatly promoted by attaching a multiphosphonate to the termini of the oligonucleotide additives and placing this ligand at the gap site. The scission fragments can be connected with foreign DNA using ligase, and the recombinant DNA expresses the corresponding protein in E. coli. No undesired side reactions (for example, depurination, deletion, insertion, oxidative damage of nucleobases and off-target scission) occur throughout the DNA treatment.

Current Status of Langmuir Monolayer Degradation of Polymeric Biomaterials

Langmuir monolayer degradation (LMD) experiments with polymers possessing outstanding biomedical application potential yield information regarding the kinetics of their hydrolytic or enzymatic chain scission under well-defined and adjustable degradation conditions. A brief review is given of LMD investigations, including the author's own work on 2-dimensional (2D) polymer systems, providing chain scission data, which are not disturbed by simultaneously occurring transport phenomena, such as water penetration into the sample or transport of scission fragments out of the sample. A knowledge-based approach for the description and simulation of polymer hydrolytic and enzymatic degradation based on a combination of fast LMD experiments and computer simulation of the water penetration is briefly introduced. Finally, the advantages and disadvantages of this approach are discussed.

Site‐Selective Blocking of PCR by a Caged Nucleotide Leading to Direct Creation of Desired Sticky Ends in The Products

In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.

Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity

The artificial restriction DNA cutter (ARCUT) method to cut double-stranded DNA at designated sites has been developed. The strategy at the base of this approach, which does not rely on restriction enzymes, is comprised of two stages: (i) two strands of pseudo-complementary peptide nucleic acid (pcPNA) anneal with DNA to form 'hot spots' for scission, and (ii) the Ce(IV)/EDTA complex acts as catalytic molecular scissors. The scission fragments, obtained by hydrolyzing target phosphodiester linkages, can be connected with foreign DNA using DNA ligase. The location of the scission site and the site-specificity are almost freely tunable, and there is no limitation to the size of DNA substrate. This protocol, which does not include the synthesis of pcPNA strands, takes approximately 10 d to complete. The synthesis and purification of the pcPNA, which are covered by a related protocol by the same authors, takes an additional 7 d, but pcPNA can also be ordered from custom synthesis companies if necessary.

Molecular weight distribution of proton irradiated polystyrene studied by diffusion experiments

MeV ion irradiation of polymers induces break-up or crosslinking of polymer chains. This leads to an ion-fluence dependent molecular weight (MW) distribution ranging from short scission fragments via larger chain clusters to the interconnected, immobile network fraction. We used diffusion experiments to obtain information about the MW distribution of the mobile chains in dependence of the ion fluence. Double layers of deuterated (dPS) and protonated (hPS) polystyrene on Si wafer substrates were irradiated by 1MeV protons and annealed at 140°C for various periods of time. The dPS diffusion depth profiles in the irradiation induced hPS network were measured by 3He nuclear reaction analysis. For calibration the strongly MW dependent diffusion coefficients of dPS chains in hPS networks were determined separately by analogous techniques. The depth profiles were fitted with MW dependent diffusion profiles convoluted with a parametrized MW distribution, and the resulting MW distribution was compared to theory.

Genetic recombination without using either restriction enzyme or PCR

We recently prepared artificial restriction DNA cutters (ARCUT) for site-selective scission of double-stranded DNA by combining Ce(IV)/EDTA complex with a pair of pseudo-complementary peptide nucleic acids (pcPNAs). Here we report an improved method for genetic recombination using ARCUT. The key point is to treat the scission fragments with nuclease S1 (a single-stranded DNA specific enzyme) and form blunt ends. By this procedure, these scission fragments are efficiently ligated with foreign DNA fragments having blunt ends, providing desired recombinant DNA in high yields. Neither restriction enzyme nor PCR amplification is required.

Manipulation of double-stranded DNA by artificial restriction enzyme composed of Ce(IV)/EDTA and PNA

Through the invasion of pseudo-complementary PNA (pePNA) to double-stranded DNA, gap-like structures were formed at predetermined sites in both strands of PBR322 plasmid DNA. These gap-like sites were selectively hydrolyzed by Ce(IV)/EDTA complex, and two designed fragments were obtained. Furthermore, the scission fragment by this artificial restriction enzyme was successfully ligated with foreign DNA.

Combination of S1 nuclease and PNA for site-selective hydrolysis of double-stranded DNA. -Comparison with the site-selective hydrolysis using Ce(IV)/EDTAY-

The potential of the combination of SI nuclease and pseudo-complementary PNA (pcPNA) for site-selective scission of double-stranded DNA has been investigated. Through strand invasion of two pcPNAs, single-stranded portions were formed in both strands of substrate DNA. In the initial stage of the enzymatic digestion, two scission fragments were obtained due to the hydrolysis at these two gap-like sites. On prolonged reactions, however, these products (as well as the substrate DNA) were further digested to smaller fragments. Under the conditions employed here, only Ce(IV)/EDTA is available for the preparation of desired fragments from double-stranded DNA.

Site-selective DNA hydrolysis by combining Ce(IV)/EDTA with monophosphate-bearing oligonucleotides and enzymatic ligation of the scission fragments.

By using two oligonucleotide additives that bear a monophosphate group at the termini through various linkers, gap structures were formed at predetermined positions in substrate DNA, and the monophosphate groups were placed at both edges of these gaps. At pH 7.0 and 37 degrees C, the phosphodiester linkages in the gap sites were efficiently and selectively hydrolyzed by Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). The linkages in the middle of the gaps were predominantly hydrolyzed. Compared with DNA scission using oligonucleotide additives that bear no terminal monophosphate, the present scission was much faster (22-fold for a 3-base gap and 14-fold for a 5-base gap) and more site selective. Introduction of one monophosphate group to either edge of the gaps was also effective for promotion of both site selectivity and scission rate. The monophosphate group(s) at the gap site recruits the Ce(IV) to the target site and magnifies the difference in intrinsic reactivity between the target site and the others. Even at higher reaction temperatures, the site selectivity remained satisfactorily high. Furthermore, the fragments formed by the site-selective scission were connected with various oligonucleotides by using DNA ligase, producing desired recombinant DNAs.

Tribochemistry in contact recording

Even though the flying height is approaching molecular dimensions, it is expected to decrease as the areal recording density is increased. This study employs a new low-velocity test to obtain ultralow flying height with sustained intermittent asperity contacts and random seek accessing in a region near the middle diameter of the disk. Sensitive surface analytical tools are employed to follow tribochemical changes. Profiles are periodically measured across the disk surface using Fourier transform infrared spectroscopy for lubricant thickness and scanning-imaging micro-ellipsometry. Zdol lubricant is removed from the tested region through evaporation of low molecular weight scission fragments. The removal rate increased with increasing chemisorbed fraction and decreasing relative humidity. The increased removal rate is attributed to increases in the Zdol relaxation time. A longer relaxation time allows more time for bond dissociation following asperity contacts. Chemisorption sites in the tested region were occupied by chemisorbed scission fragments with a carboxylic acid end group. Amine and sodium salts of the carboxylic acid were detected on the tested regions of Zdol lubricated, and unlubricated, overcoats. The presence of tribochemically generated carboxylic acid groups on Zdol and overcoat carbon explain the increased water adsorption measured on the tested regions.

Model experiment on the protection effect in polymers: radiolysis of liquid squalane in the presence and absence of additives

A model experiment to clarify the elementary processes in aliphatic polymers irradiated under vacuum has been carried out. Liquid squalane has been selected as the model of branched polyethylene, ethylene-propylene copolymers or polypropylene. The main gaseous product is H 2. The presence of methyl side groups increases the probability of main chain scission, and side chain scission produces methane as the main hydrocarbon gas product. The prominence of main chain scission and the chemical structure of the parent molecule are believed to be the reasons for formation of less oligomeric products. On the other hand, they promote the combination of the scission fragment with the parent radicals to produce intermediate compounds. In the presence of aromatic additives which have a lower ionization potential and lower excited state energy level than the squalane itself, energy and charge transfer to the added molecules take place and both solvent decomposition and product formation including H 2 evolution are significantly reduced. Due to these transfer processes, the added molecules react with squalane molecules to produce adduct compounds which are clearly observed from the mass analysis. A further inspection of the mass spectra shows that scavenging of hydrogen atoms occurs through hydrogenation of the benzene rings in the adducts. Since the adduct compounds of aromatic additives consume more hydrogen atoms than those of hydroaromatic additives, aromatic compounds reduce H 2 evolution more effectively than do hydroaromatic compounds. Since main chain scission is not affected by any of the additives, it is suggested that this scission process directly originates from the decomposition of the excited cations of squalane. The G-values in the radiolysis of liquid alkanes and base polymers are also compared.

Model‐filled polymers. II. Stability of polystyrene beads in a polystyrene matrix

AbstractThermomechanical degradation, as a result of mixing at 175°C during the preparation of polystyrene composites containing monodisperse (0.45μm) crosslinked polystyrene beads, often leads to a uniform decrease in rheological properties on increasing the concentration or on reducing the crosslink density of the beads. The extent of such degradation depends on the molecular weight of polystyrene and on the crosslink density of the polystyrene beads. Scission fragments from decomposed beads may induce free radical degradation of the polystyrene matrix. Degradation during mixing of the beads and the matrix is suppressed by mixing crosslinked beads in a polystyrene solution, and removing the solvent, or by melt mixing after adding 0.2% BHT. Polystyrene beads and composites show thermally induced weight loss on heating above 330°C in nitrogen or above 250°C in air. Networks of increased crosslink density are of enhanced thermal stability on heating in the absence of shear.

Base sequence damage in DNA from X-irradiated monkey CV-1 cells.

Two kinds of 3'-ends were detected in DNA scission fragments of highly repetitive primate component alpha DNA which were isolated from irradiated monkey CV-1 cells. The fragments' 3'-ends were characterized by 5'-32P-end labelling the DNA, followed by examination in high-resolution polyacrylamide gels under denaturing conditions. Hydrolysis of the labelled fragments' termini with exonuclease III of E. coli or by the 3'-phosphatase activity of T4 polynucleotide kinase generated a third, slowest migrating species in each mobility size class. Reference to mobility size class standards makes it highly probable that the fragment ends generated by X-rays in cells are 3'-phosphoryl and 3'-phosphoglycolate, and that they are converted to slower migrating fragments with 3'-OH ends, similar to results obtained with DNA irradiated in water (Henner et al. 1982, 1983 a, b). Densitometer measurements of gel autoradiograms showed that X-ray induction of DNA fragments with 3'-phosphoryl and 3'-phosphoglycolate ends was dose-dependent over a range 100-900 Gy. In CV-1 cells the frequency of single-strand breaks in alpha DNA was 8.6 x 10(-7) breaks/nt/Gy. The two kinds of ends disappeared in post-radiation incubation with a half-time of 1.6 h. These results provide a new means to study X-ray damage and repair of specific sequences in animal cells.

Comparison of Radical and Nonradical Processes in the Condensed-Phase Radiolysis of n-Hexadecane

The effects of state, temperature, and chemically reactive solutes on the radiolysis of n-hexadecane in condensed phases have been examined. All product yields are reduced by radical scavengers in liquid-state n-hexadecane, but only dotriacontane isomers are reduced in the solid. Iodine acts as a chemical scavenger and is more effective than 2-methylpentene−1. A hot-hydrogen-atom mechanism, together with molecular elimination, accounts for the formation of most of the hydrogen in the irradiation of n-hexadecane. Low-molecular-weight products are formed entirely by molecular reactions in the solid-state radiolysis and, about equally, via molecular and radical processes in the liquid. Intermediate-molecular-weight products in the liquid radiolysis are 80% from combinations of radicals from chain scission with various hexadecyl radicals. The remaining 20% in the liquid and the total intermediate yield in the solid are from nonradical processes. The combination of hexadecyl radicals yields at least 40% and 70% of the dotriacontane isomers in the radiolysis of pure n-hexadecane in the solid and liquid state, respectively. The remaining dimer may also be due to radicals which are formed in pairs, but ion—molecule reactions cannot be excluded. Temperature dependence of all products is small. Cage recombination of scission fragments and crosslinking of proximate radical pairs is enhanced in the solid state as compared with the liquid. Internal ···CH2–CH2··· rupture is equally probable at all positions, and seven times as likely as terminal CH3–CH2··· scission. Methyl C–H rupture is 40% as probable per bond as methylene C–H rupture.