Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.     

Synthesis of ferrocenyl-bearing dendrimers with a resorcinarene core

A series of ferrocenyl ended dendrons containing π-conjugated systems were obtained using Wittig and Heck reactions. The dendrons were attached to eight functionalized resorcinarenes via Williamson reaction obtaining high molecular weight dendrimers. No electronic communication between metal centers was observed by cyclic voltammetry. All the dendrimers were characterized by ¹H, ¹³C NMR, FTIR, UV-Vis, MALDI-TOF, elemental analyses, and electrochemical studies.     

Polycationic lipophilic-core dendrons as penetration enhancers for the oral administration of low molecular weight heparin

Two polycationic lipophilic-core carbohydrate-based dendrons 2a-b and five polycationic lipophilic-core peptide dendrons 3-6, containing four arginine or lysine terminal residues, were synthesized and then tested in rats as penetration enhancers for the oral delivery of low molecular weight heparin. Better results were obtained with dendrons containing terminal lysine residues than terminal arginine. A significant anti-factor Xa activity was obtained when low molecular weight heparin was coadministered with dendron 5.     

Molecular mechanisms of action of dendrons, containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of the adenylyl cyclase signaling systems

For the aims of studying molecular mechanisms of functioning of adenylyl cyclase signaling systems (ACS), we investigated the influence of synthetic polycationic peptides of the star-like structure (dendrons), containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of ACS components in smooth muscles of the mollusc Anodonta cygnea and in rat skeletal muscles. It has been shown that the following peptides (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx(= epsilon-aminohexanoic acid)-Cys(Acm), referred to as peptide I, (Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys(Acm) (peptide II), [(Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide III), and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide IV) inhibit in a dose-dependent manner the adenylyl cyclase (AC) activity stimulated by both nonhormanal agents (GppNHp and forskolin) and hormones, such as serotonin (mollusc) and isoproterenol (rat). Peptides III and IV (tetrameric dendrons) were most effective in comparison with peptides I and II (dimeric dendrons). The AC activity stimulated by hormones and forskolin was most sensitive to the action of dendrons. All dendrons stimulated GTP-binding activity of G-proteins: dimeric dendrons were most effective at 10(-5) M concentration, whereas tetrameric dendrons at 10(-6) M. In the presence of dendrons, the affinity of beta-antagonist [3H]-dihydroalprenolol to P-adrenergic receptor in rat muscle mem- branes was unchanged. At the same time, the affinity of beta-agonist isoproterenol to the receptor decreased, and no shift to the right was observed on the curve of isoproterenol-induced [3H]-dihydroalprenolol displacement in the presence of GTP. The obtained data show the disturbance of the coupling between the receptor and G-protein, which is the main reason of dendron inhibitory action on AC stimulation by hormones. Besides, these data demonstrated that hormones could disturb the functional activity of AC, i.e. a catalytic component of ACS.     

Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection.

Native and modified synthetic oligonucleotides were purified by reversed-phase HPLC using volatile ion-pairing mobile phases. Purification of 10-90 nmol of oligonucleotides in a single injection was demonstrated using a 4.6 x 75-mm HPLC column packed with porous 2.5 microm C18 sorbent. Separation of target products from N-1 failure fragments was achieved for oligonucleotides in the 4- to 60-mer size range. We employed a combination of absorbance and mass spectrometry detection to identify by-products of oligonucleotide synthesis. This method was also employed for analysis and purification of fluorescently labeled oligonucleotides.     

Determination of oligonucleotide molecular masses by MS-MALDI

MALDI mass spectrometry (MS) of 14- to 42-mer homogeneous oligonucleotides and their mixtures was carried out using a Vision 2000 instrument (Thermo BioAnalysis, Finnigan, United States). Conditions for the determination of oligonucleotide molecular masses were optimized by applying various matrices and operation modes. The most reproducible results with minimal uncontrolled decomposition of the oligonucleotides including their apurinization during the MALDI MS registration were obtained using 2,4,6-trihydroxyacetophenone as a matrix instead of 3-hydroxypicolinic acid, usually employed in the mass spectrometry of oligonucleotides. Our approach allows the determination of molecular masses of oligonucleotides obtained by chemical synthesis and the evaluation of their component composition and purity. It was applied to the mass spectrometric analysis of oligonucleotides containing a 3'-(methyl-C-phosphonate) group or a modified 1,N6-ethenodeoxyadenosine unit.     

Development of LNA oligonucleotide–PCR clamping technique in investigating the community structures of plant-associated bacteria

Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.     

Development of a Quantitative Polyacrylamide Gel Electrophoresis Analysis Using a Multichannel Radioactivity Counter for the Evaluation of Oligonucleotide-Bound Drug Carrier

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of³³P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.     

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.     

Novel monodisperse PEG-dendrons as new tools for targeted drug delivery: synthesis, characterization and cellular uptake

Dendrimers, dendrons, and hyperbranched polymers are gaining popularity as novel drugs, imaging agents, and drug delivery systems. They present advantages of well-defined molecular weight, multivalent surfaces, and high drug carrying capacity. Moreover, it is emerging that such architectures can display unique endocytic properties. As poly(ethylene glycol) (PEG) is widely used for protein and drug conjugation, the aim of this study was for the first time to synthesize novel, branched PEG-based architectures, to define their cytotoxicity and, via preparation of Oregon green (OG) conjugates define the effect of structure on their cellular uptake. Five PEG-based dendrons were synthesized using monodisperse Fmoc-amino PEG propionic acid (M(w) = 840) as a monomer, and cadaverine, tris(2-aminoethyl)amine or lysine as the branching moieties. These were diamino,bisPEG (M(w) = 1300); triamino,trisPEG (Mw = 1946); tetraamino,tetraPEG (M(w) = 3956); monocarboxy,diamino,bisPEG (M(w) = 1346); and monocarboxy,tetraamino,tetraPEG (M(w) = 3999). These products had NH(2) or both NH(2) and COOH terminal groups and the identity was verified by amino group analysis and ESI-TOF mass spectroscopy. Purity was determined by HPLC. Representative structures were not toxic towards an endothelial-like cell line (ECV304) at concentrations up to 4 mg/mL (over 72 h). At 37 degrees C, all of the OG-labeled PEG dendrons showed progressive uptake by ECV304 cells, but tetraamino,tetraPEG showed the greatest rate of internalization over the first 20 min. Cellular uptake was inhibited at 4 degrees C, and PEG dendron localization to perinuclear vesicles was confirmed by fluorescence microscopy. These well-defined novel architectures have potential for further development as targetable drug delivery systems or tools for construction of structurally defined modified surfaces.     

Dual specificity antibodies using a double-stranded oligonucleotide bridge.

The covalent conjugation of oligonucleotides to antibody Fab' fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics.     

A hybridization procedure for the isolation of specific RNA segments applied to the analysis of bacteriophage Qbeta RNA.

A method for the isolation of RNA fragments originating from defined regions of bacteriophage Qbeta RNA minus strands is described. Large RNase T1 oligonucleotides were isolated on a preparative scale from Qbeta RNA. The nucleotide sequences (13 to 26 nucleotides) and map positions of these oligonucleotides were known from previous work (Billeter, M. A. (1978) J. Biol. Chem. 253, 8381-8389). After addition of AMP residues (50 in the average) using terminal adenylate transferase, these pure oligonucleotides were hybridized to 32P-labeled Qbeta RNA minus strands synthesized in vitro. Fragments in the size range of 100 to 500 nucleotides were then generated by partial digestion with RNase T1. Fragments hybridized to such oligonucleotides were recovered by chromatography on poly(U)-Sephadex and then resolved according to their size by polyacrylamide gel electrophoresis. The specificity and reproducibility of the method as well as its suitability for the sequence analysis of Qbeta RNA was verified by using in particular a linker oligonucleotide derived from a Qbeta RNA region near the 3' end. The sequence catalogues of the RNase T1 and RNase A oligonucleotides of two fragments isolated in this way, 202 and 310 nucleotides in length, were established and all fragments isolated were shown to contain a sequence complementary to the linker oligonucleotide.     

The addition of low numbers of 3' thymine bases can be used to improve the hybridization signal of oligonucleotides for use within arrays on nylon supports

Oligonucleotide arrays can be used for the analysis of microbial nucleic acid. The addition of high numbers of dTTP to the 3' ends of oligonucleotides using terminal transferase has been shown to facilitate membrane binding. This paper demonstrates low numbers of thymine bases added to the 3' end of oligonucleotides during synthesis can improve hybridisation signal intensity where the signal seen with the unmodified oligonucleotides is poor. Thus, the addition of variable numbers of thymine bases to different oligonucleotides allows the production of oligonucleotide arrays producing strong interpretable hybridisation signals.     

Synthesis of symmetrical and unsymmetrical PAMAM dendrimers by fusion between azide- and alkyne-functionalized PAMAM dendrons

A new convergent synthetic method for the synthesis of PAMAM dendrimers has been developed. The fusion between propargyl-functionalized PAMAM dendrons and azido-functionalized PAMAM dendrons via the Cu(I)-catalyzed Huisgen [2 + 3] dipolar cycloaddition reaction (click chemistry) of an alkyne and an azide leads to the formation of symmetric PAMAM dendrimers in high yields. Furthermore, the coupling reactions between the different generation dendrons afford the size-differentiated unsymmetrical PAMAM dendrimers.     

Oligonucleotide analysis by anion exchange HPLC.

The ability to resolve and purify synthetic oligonucleotides by high performance anion exchange chromatography was evaluated using two wide pore polymeric HPLC matrices. The materials used are rigid macroporous copolymers which have a fully quaternised polyethyleneimine coating to provide a strong anion exchange, quaternary amine, functionality. Oligomers of poly(rA), poly(rC) and RNA produced by alkaline hydrolysis of the polymers were chromatographed to evaluate the selectivity of the system prior to the analysis of synthetic oligonucleotides produced using a commercial oligonucleotide synthesizer.     

Attachment of oligonucleotide probes to poly carbodiimide-coated glass for microarray applications

Oligonucleotide-based DNA microarrays are becoming increasingly useful tools for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Here, we present a method that permits the manufacture of microarrays from non-modified oligonucleotides on a poly carbodiimide-coated glass surface by UV-irradiation. The use of UV-irradiation facilitates an increase in the level of signal intensity, but it does not affect signal discrimination by the oligonucleotides immobilized on the surface. The signal intensity obtained for an array fabricated using non-modified oligonucleotides with UV-irradiation is ~7-fold greater than that without UV-irradiation. The detection of SNPs was tested to ascertain whether this technique could discriminate specific hybridization signals without causing significant UV-irradiation-induced damage to the immobilized oligonucleotides. We found that this immobilization method provides greater hybridization signals and a better match/mismatch ratio of SNPs than do the established aminosilane techniques. Application of this technology to manufacturing DNA microarrays for sequence analysis is discussed.     

Quantitative Parametrs of Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template

Abstract

The cooperative interactions of oligonucleotides on the complementary template were studied using the quantitative analysis of the template alkylation with the oligonucleotides bearing covalently attached 4-[N-(2-chloroethyl)-N-methylamino]benzyl group at 5′-end. The influence of the mismatched nucleotides and the stabilizing N-(2-hydroxyethyl)phenazinium group at the 5′- and 3′-ends of the oligonucleotides on the parameters of cooperativity was evaluated.     

A unitary hypothesis of mind-brain interaction in the cerebral cortex

A brief introduction to the brain-mind problem leads on to a survey of the neuronal structure of the cerebral cortex. It is proposed that the basic receptive units are the bundles or clusters of apical dendrites of the pyramidal cells of laminae V and III-II as described by Fleischhauer and Peters and their associates. There are up to 100 apical dendrites in these receptive units, named dendrons. Each dendron would have an input of up to 100,000 spine synapses. There are about 40 million dendrons in the human cerebral cortex. A study of the influence of mental events on the brain leads to the hypothesis that all mental events, the whole of the World 2 of Popper, are composed of mental units, each carrying its own characteristic mental experience. It is further proposed that each mental unit, named psychon, is uniquely linked to a dendron. So the mind-brain problem reduces to the interaction between a dendron and its psychon for all the 40 million linked units. In my 1986 paper (Proc. R. Soc. Lond. B 227, 411-428) on the mind-brain problem, there was developed the concept that the operation of the synaptic microsites involved displacement of particles so small that they were within range of the uncertainty principle of Heisenberg. The psychon-dendron interaction provides a much improved basis for effective selection by a process analogous to a quantal probability field. In the fully developed hypothesis psychons act on dendrons in the whole world of conscious experiences and dendrons act on psychons in all perceptions and memories. It is shown how these interactions involve no violation of the conservation laws. There are great potentialities of these unitary concepts, for example as an explanation of the global nature of a visual experience from moment to moment. It would seem that there can be psychons not linked to dendrons, but only to other psychons, creating what we may call a psychon world.     

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named “Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides” were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability.