Design of three-dimensional domain-swapped dimers and fibrous oligomers

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 Å resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.

Inhibition of telomerase by 2′-O-(2-methoxyethyl) RNA oligomers: effect of length, phosphorothioate substitution and time inside cells

2′-O-(2-methoxyethyl) (2′-MOE) RNA possesses favorable pharmocokinetic properties that make it a promising option for the design of oligonucleotide drugs. Telomerase is a ribonucleoprotein that is up-regulated in many types of cancer, but its potential as a target for chemotherapy awaits the development of potent and selective inhibitors. Here we report inhibition of human telomerase by 2′-MOE RNA oligomers that are complementary to the RNA template region. Fully complementary oligomers inhibited telomerase in a cell extract with IC50 values of 5–10 nM at 37°C. IC50 values for mismatch-containing oligomers varied with length and phosphorothioate substitution. After introduction into DU 145 prostate cancer cells inhibition of telomerase activity persisted for up to 7 days, equivalent to six population doublings. Inside cells discrimination between complementary and mismatch-containing oligomers increased over time. Our results reveal two oligomers as especially promising candidates for initiation of in vivo preclinical trials and emphasize that conclusions regarding oligonucleotide efficacy and specificity in cell extracts do not necessarily offer accurate predictions of activity inside cells.

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Synthesis and Characterization of Lactic Acid Oligomers: Evaluation of Performance as Poly(Lactic Acid) Plasticizers

The use of fully bio-based and biodegradable materials for massive applications, such as food packaging, is an emerging tendency in polymer research. But the formulations proposed in this way should preserve or even increase the functional properties of conventional polymers, such as transparency, homogeneity, mechanical properties and low migration of their components to foodstuff. This is not always trivial, in particular when brittle biopolymers, such as poly(lactic acid) (PLA), are considered. In this work the formulation of innovative materials based on PLA modified with highly compatible plasticizers, i.e. oligomers of lactic acid (OLAs) is proposed. Three different synthesis conditions for OLAs were tested and the resulting additives were further blended with commercial PLA obtaining transparent and ductile materials, able for films manufacturing. These materials were tested in their structural, thermal and tensile properties and the best formulation among the three materials was selected. OLA with molar mass (Mn) around 1,000 Da is proposed as an innovative and fully compatible and biodegradable plasticizer for PLA, able to replace conventional plasticizers (phthalates, adipates or citrates) currently used for films manufacturing in food packaging applications.

Chemical synthesis of isotopically labelled (M+4) purine nucleosides and their incorporation into DNA oligomers

Development of accurate and sensitive analytical methods to measure the level of biomarkers, such as 8-oxo-guanine or its corresponding nucleoside, 8-oxo-2’-deoxyguanosine, has become imperative in the study of DNA oxidative damage in vivo. Of the most promising techniques, HPLC-MS/MS, has many attractive advantages. Like any method that employs the MS technique, its accuracy depends on the use of multiply, isotopically-labelled internal standards. This project is aimed at making available such internal standards. The first task was to synthesise the multiply, isotopically-labelled bases (M+4) guanine and (M+4) 8-oxo-guanine. Synthetic routes for both (M+4) guanine and (M+4) 8-oxo-guanine were designed and validated using the unlabelled compounds. The reaction conditions were also optimized during the “dry runs”. The amination of the 4-hydroxy-2,6-dichloropyrimidine, appeared to be very sensitive to the purity of the commercial [15]N benzylamine reagent. Having failed, after several attempts, to obtain the pure reagent from commercial suppliers, [15]N benzylamine was successfully synthesised in our laboratory and used in the first synthesis of (M+4) guanine. Although (M+4) bases can be, and indeed have been used as internal standards in the quantitative analysis of oxidative damage, they can not account for the errors that may occur during the early sample preparation stages. Therefore, internal standards in the form of nucleosides and DNA oligomers are more desirable. After evaluating a number of methods, an enzymatic transglycolization technique was adopted for the transfer of the labelled bases to give their corresponding nucleosides. Both (M+4) 2-deoxyguanosine and (M+4) 8-oxo-2’-deoxyguanosine can be purified on micro scale by HPLC. The challenge came from the purification of larger scale (>50 mg) synthesis of nucleosides. A gel filtration method was successfully developed, which resulted in excellent separation of (M+4) 2’-deoxyguanosine from the incubation mixture. The (M+4) 2’-deoxyguanosine was then fully protected in three steps and successfully incorporated, by solid supported synthesis, into a DNA oligomer containing 18 residues. Thus, synthesis of 8-oxo-deoxyguanosine on a bigger scale for its future incorporation into DNA oligomers is now a possibility resulting from this thesis work. We believe that these internal standards can be used to develop procedures that can make the measurement of oxidative DNA damage more accurate and sensitive.

Dielectric relaxation of polysiloxanes and kerr effect of p-phenylene vinylene oligomers

The dielectric relaxation behaviour of a series of cyclic and linear poly(dimethylsiloxanes) with overline nn in the range 28 to 99 has been studied, as a function of temperature (142.0K-157.5K) and frequency (12-105Hz). Activation energies for the -relaxation process, Davidson-Cole empirical distribution factors, , and mean-square dipole moments per repeat unit, < 2> , have been calculated. Differences in values of H_act reflected restricted dipolar rotation for the cyclic structures, compared to the linear structures, over the range of molecular weights studied. The dielectric relaxation behaviour of a series of linear oligomers of methyl phenyl siloxane, with n in the range 4 to 10, a series of linear fractions of poly(methyl phenyl siloxane), with overline n_n in the range 31 to 1370, and a cyclic oligomer of mehyl phenyl siloxane, with n = 10, has been studied as a function of temperature (155.5K-264.0K) and frequency (12-105Hz). Activation energies for the -relaxation process, Davidson-Cole and Cole-Cole empirical distribution factors, and , respectively, and mean-square dipole moments per repeat unit have been calculated. The reduced flexibility of short methyl phenyl siloxane chains, compared to dimethyl siloxane chains, was apparent from a comparison of dipole moment ratios. The dilectric relaxation behaviour of poly(methyl hydrogen siloxane) and poly(n-hexyl methyl siloxane) has been studied as a function of temperature and frequency. A polysiloxane liquid crystal has been synthesised and its dielectric relaxation behaviour has been studied, as a function of temperature and frequency, in the liquid crystalline phase and below T_g. Poly(p-phenylene vinylene) and related oligomers have been synthesised and characterised by a variety of experimental techniques. The Kerr effect of two oligomeric fractions, in solution in PPG 2025, has been measured. The electrical conductivities of the undoped and I_2-doped polymer and oligomers have been measured.

The dawn of the RNA World:toward functional complexity through ligation of random RNA oligomers

A main unsolved problem in the RNA World scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers obtained by random polymerization on mineral surfaces. A number of computational studies have shown that the structural repertoire yielded by that process is dominated by topologically simple structures, notably hairpin-like ones. A fraction of these could display RNA ligase activity and catalyze the assembly of larger, eventually functional RNA molecules retaining their previous modular structure: molecular complexity increases but template replication is absent. This allows us to build up a stepwise model of ligation- based, modular evolution that could pave the way to the emergence of a ribozyme with RNA replicase activity, step at which information-driven Darwinian evolution would be triggered. Copyright © 2009 RNA Society.

Pore confinement effects and stabilization of carbon nitride oligomers in macroporous silica for photocatalytic hydrogen production

An ordered macroporous host (mac-SiO2) has been used to prevent aggregation of layered photocatalysts based on carbon nitride. Using typical carbon nitride synthesis conditions, cyanamide was condensed at 550 °C in the presence and absence of mac-SiO2. Condensation in the absence of mac-SiO2 results in materials with structural characteristics consistent with the carbon nitride, melon, accompanied by ca. 2 wt% carbonization. For mac-SiO2 supported materials, condensation occurs with greater carbonization (ca. 6 wt%). On addition of 3 wt% Pt cocatalyst photocatalytic hydrogen production under visible light is found to be up to 10 times greater for the supported composites. Time-resolved photoluminescence spectroscopy shows that excited state relaxation is more rapid for the mac-SiO2 supported materials suggesting faster electron-hole recombination and that supported carbon nitride does not exhibit improved charge separation. CO2 temperature programmed desorption indicates that enhanced photoactivity of supported carbon nitride is attributable to an increased surface area compared to bulk carbon nitride and an increase in the concentration of weakly basic catalytic sites, consistent with carbon nitride oligomers.

Hybrid Diphenylalkyne-Dipeptide Oligomers Induce Multistrand β-Sheet Formation

Functionalized diphenylalkynes provide a template for the presentation of protein-like surfaces composed of multistrand β-sheets. The conformational properties of three-, four-, and seven-stranded systems have been investigated in the solid- and solution-state. This class of molecule may be suitable for the mediation of therapeutically relevant protein-protein interactions.

Synthesis and characterization of sodalite–polyimide nanocomposite membranes

Nanocomposite membranes are fabricated from sodalite nanocrystals (Sod-N) dispersed in BTDA-MDA polyimide matrices and then characterized structurally and for gas separation. No voids are found upon investigation of the interfacial contact between the inorganic and organic phases, even at a Sod-N loading of up to 35 wt.%. This is due to the functionalization of the zeolite nanocrystals with amino groups (==Si_(CH3)(CH2)3NH2), which covalently link the particles to the polyimide chains in the matrices. The addition of Sod-N increases the hydrogen-gas permeability of the membranes, while nitrogen permeability decreases. Overall, these nanocomposite membranes display substantial selectivity improvements. The sodalite–polyimide membrane containing 35 wt.% Sod-N has a hydrogen permeability of 8.0 Barrers and a H2/N2 ideal selectivity of 281 at 25 C whereas the plain polyimide membrane exhibits a hydrogen permeability of 7.0 Barrers and a H2/N2 ideal selectivity of 198 at the same testing temperature.

Fumaric acid monoethyl ester-functionalized poly(D,L-Lactide)/N-vinyl-2- pyrrolidone Resins for the preparation of tissue engineering scaffolds by stereolithography

Polymer networks were prepared by photocross-linking fumaric acid monoethyl ester (FAME) functionalized, three-armed poly(D,L-lactide) oligomers using Af-vinyl-2-pyrrolidone (NVP) as diluent and comonomer. The use of NVP together with FAME-functionalized oligomers resulted in copolymerization at high rates, and networks with gel contents in excess of 90 were obtained. The hydrophilicity of the poly(D,L-lactide) networks increases with increasing amounts of NVP, networks containing 50 wt of NVP absorbed 40 of water. As the amount of NVP was increased from 30 to 50 wt , the Young's modulus after equilibration in water decreased from 0.8 to 0.2 GPa, as opposed to an increase from 1.5 to 2.1 GPa in the dry state. Mouse preosteoblasts readily adhered and spread onto all prepared networks. Using stereolithography, porous structures with a well-defined gyroid architecture were prepared from these novel materials. This allows the preparation of tissue engineering scaffolds with optimized pore architecture and tunable material properties.

A poly(D,L-lactide) resin for the preparation of tissue engineering scaffolds by stereolithography

Porous polylactide constructs were prepared by stereolithography, for the first time without the use of reactive diluents. Star-shaped poly(D,L-lactide) oligomers with 2, 3 and 6 arms were synthesised, end-functionalised with methacryloyl chloride and photocrosslinked in the presence of ethyl lactate as a non-reactive diluent. The molecular weights of the arms of the macromers were 0.2, 0.6, 1.1 and 5 kg/mol, allowing variation of the crosslink density of the resulting networks. Networks prepared from macromers of which the molecular weight per arm was 0.6 kg/mol or higher had good mechanical properties, similar to linear high molecular weight poly(D,L-lactide). A resin based on a 2-armed poly(D,L-lactide) macromer with a molecular weight of 0.6 kg/mol per arm (75 wt%), ethyl lactate (19 wt%), photo-initiator (6 wt%), inhibitor and dye was prepared. Using this resin, films and computer-designed porous constructs were accurately fabricated by stereolithography. Pre-osteoblasts showed good adherence to these photocrosslinked networks. The proliferation rate on these materials was comparable to that on high molecular weight poly(D,L-lactide) and tissue culture polystyrene.

Development of a PDLLA-based stereolithography resin for making tissue engineering scaffolds

The use of porous structures as tissue engineering scaffolds imposes high demands on the pore architecture. Stereolithography is a rapid prototyping method based on photo-polymerisation, that can be utilised to make 3D constructs with high spatial control. In this study, biodegradable resins were developed that can find application in stereolithography. Poly(D,L-lactide) (PDLLA) oligomers were synthesised and functionalised with methacrylate end-groups. By mixing the resulting macromers with a diluent, photo-initiator and inhibitor, lowviscosity resins were obtained that were photocrosslinked to yield stiff and strong degradable poly(lactide) networks. Also, porous scaffolds were fabricated on a stereolithography apparatus (SLA) from a nondegradable resin.

Properties of photo-cured poly(D,L-lactide) networks

Poly(D,L-lactide) is a degradable polymer with a long history of use in medical applications. It is strong and stiff and degrades over the course of months into lactic acid, a body-own substance. In the field of tissue engineering it is commonly used to fabricate scaffolds. Stereolithography is a high resolution rapid prototyping technique by which designed 3D objects can be built using photo-initiated radical polymerisations. Poly(D,Llactide) (PDLLA) networks can be obtained by photopolymerisation of oligomers functionalised with unsaturated groups. In this work, PDLLA oligomers of varying architectures (arm lengths, numbers of arms) were synthesised and end-functionalised with methacrylate groups. These macromers were photo-crosslinked in solution to yield PDLLA networks of different architectures. The influence of the network architecture on its physical properties was studied.

Reinforced hydrogels for silicone copolymer delivery for scar remediation

Hypertrophic scars are formed by collagen overproduction in wounded areas and often occur in victims of severe burns. There are several methods for hypertrophic scar remediation and silicone gel therapy is one of the more successful methods. Research by others has shown that the activity of these gels may be due to migration of amphiphilic silicone oligomers from the gel and into the dermis, down-regulating production of collagen by fibroblasts. Normal silicone oil (PDMS) does not produce the same effect on fibroblasts. The main purpose of this project is the introduction of a particular amphiphilic silicone rake copolymer into an appropriate network which can absorb and release the silicone copolymer on the scarred area. Hydrogels are polymeric crosslinked networks which can swell in water or a drug solution, and gradually release the drug when applied to the skin. The application of gel enhances the effectiveness of the therapy, reduces the period of treatment and can be comfortable for patients to use. Polyethylene glycol (PEG) based networks have been applied in this research, because the amphiphilic silicone rake copolymer to be used as a therapy has polyethylene oxide (PEO) as a side chain. These PEO side chains have very similar chemical structure to a PEG gel chain so enhancing both the compatibility and the diffusion of the amphiphilic silicone rake copolymer into and out of the gel. Synthesis of PEG-based networks has been performed by two methods: in situ silsesquioxane formation as crosslink with a sol-gel reaction under different conditions and UV curing. PEG networks have low mechanical properties which is a fundamental limitation of the polymer backbone. For mechanical properties enhancement, composite networks were synthesized using nano-silica with different surface modification. The chemical structure of in situ silsesquioxane in the dry network has been examined by Solid State NMR, Differential Scanning Calorimetry (DSC) and swelling measurements in water. Mechanical properties of dry networks were tested by Dynamic Mechanical Thermal Analysis (DMTA) to determine modulus and interfacial interaction between silica and the network. In this way a family of self-reinforced networks has been produced that have been shown to absorb and deliver the active amphiphilic silicone- PEO rake copolymer.