An Update on Protection of 5'-Hydroxyl Functions of Nucleosides and Oligonucleotides.

The synthesis of natural and chemically modified nucleosides and oligonucleotides is in great demand due to its increasing number of applications in diverse areas of research. These include tools for diagnostics and proteomics, research reagents for molecular biology, probes for functional genomics, and the design, discovery, development, and manufacture of new therapeutics. The likelihood of success in synthesizing these molecules is often dependent on the correct choice of a protection strategy to block the 5'-hydroxyl group of a carbohydrate moiety, nucleoside, or oligonucleotide. This topic was reviewed extensively in the year 2000. The purpose of this article is to complement and update the original review with recently published methodologies recommended for the protection and deprotection of the 5'-hydroxyl group. © 2024 Wiley Periodicals LLC.

Differences in uptake, localization, and processing of PNAs modified by COX VIII pre-sequence peptide and by triphenylphoshonium cation into mitochondria of tumor cells.

Two approaches to target PNAs (peptide nucleic acids) into mitochondria of HeLa cells were compared. In the first, PNA was modified with the lipophilic cation TPP. TPP-PNA accumulated rapidly within mitochondria driven by the membrane potential. It was found to be associated mainly with the mitochondrial membranes. In the second approach the COX VIII pre-sequence peptide was added to the PNA resulting in slow uptake of the peptide-PNA into the mitochondrial matrix. Whereas the amount of the uptake was lower, peptide-PNA was processed intramitochondrially in contrast to the TPP-PNA. Using the Chariot system to cross the cell membrane of HeLa cells, the uptake of peptide-PNA into the mitochondria was demonstrated. If a matrix localization of the free PNA is a pre-requisite for the PNA interaction with mitochondrial DNA, the coupling PNA with an appropriate peptide seems to be the better strategy.

Genetically engineered clostridial C2 toxin as a novel delivery system for living mammalian cells.

The C2 toxin of Clostridium botulinum is a binary bacterial protein toxin, comprising the enzyme component C2I and the separate binding/translocation component C2IIa. C2IIa mediates the transport of C2I into the host cell cytosol. The N-terminal domain of C2I (C2IN) is enzymatically inactive but essential for C2IIa-mediated internalization of C2I. Here, we exploit the C2IIa/C2IN system to generate a recombinant C2IN-streptavidin fusion protein allowing for the delivery of biotinylated molecules into the cytosol of mammalian cells. C2IN-streptavidin overproduced in E. coli was affinity-purified and capable of binding biotinylated proteins in a concentration-dependent manner. Real-time surface plasmon resonance confirmed the biotin-mediated interaction yielding a K(D)-value of approximately 0.75 muM. Internalization of C2IN-streptavidin into the cytosol of epithelial cells and macrophages was demonstrated by immunoblot analysis and confirmed by confocal microscopy. Cell viability studies showed no cytotoxic effects of the novel transporter. Furthermore, Vero cells treated with biotin-fluorescein or biocytin-Alexa488 as model cargo displayed a specific C2IN-streptavidin/C2IIa-dependent uptake, providing proof-of-principle for the functionality of this novel delivery system.

Introduction: array technology - an overview.

Microarray technology has its roots in high-throughput parallel synthesis of biomacromolecules, combined with combinatorial science. In principle, the preparation of arrays can be performed either by in situ synthesis of biomacromolecules on solid substrates or by spotting of ex situ synthesized biomacromolecules onto the substrate surface. The application of microarrays includes spatial addressing with target (macro) molecules and screening for interactions between immobilized probe and target. The screening is simplified by the microarray format, which features a known structure of every immobilized library element. The area of nucleic acid arrays is best developed, because such arrays are allowed to follow the biosynthetic pathway from genes to proteins, and because nucleic acid hybridization is a most straightforward screening tool. Applications to genomics, transcriptomics, proteomics, and glycomics are currently in the foreground of interest; in this postgenomic phase they are allowed to gain new insights into the molecular basis of cellular processes and the development of disease.

Immobilization and hybridization of oligonucleotides on maleimido-terminated self-assembled monolayers.

Maleimido-terminated self-assembled monolayers were prepared via a one-step reaction of a maleimido-functionalized trichlorosilane with an oxide-covered surface. Through conjugate addition, thiol-tagged DNA was immobilized on these maleimido-functionalized self-assembled monolayers, and these immobilized oligonucleotides were further hybridized with complementary strands. The surface concentrations of oligonucleotides were quantified with radioactivity measurements. The surface concentrations of immobilized oligonucleotides increase in a nonlinear manner with increasing molar fraction of maleimido moieties on the surface and achieve high values of 1.6 (+/-0.6) x 10(13) oligonucleotides/cm2 if monolayers having full coverage of maleimido groups are used. High hybridization efficiencies of complementary strands (approximately 75%) are obtained even if the surface concentration of immobilized strands exceeds a value of 5.0 x 10(12) oligonucleotides/cm2. Furthermore, five cycles of denaturation and rehybridization result in only a 4% loss of the immobilized DNA after each run.

Ultrathin functional star PEG coatings for DNA microarrays.

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.

Enzymatic and hybridization properties of oligonucleotide analogs containing novel phosphoramidate internucleotide linkages.

In line with the paradigm, that antisense oligonucleotides should contain minimal structural modifications, in order to minimize the risk of toxicity and antigenicity, we describe here the preparation and the properties of oligonucleotides modified to contain, in addition to phosphodiester bonds, a small number of phosphoramidate internucleotide linkages substituted with aminoethoxyethyl groups in order to convey protection against exo- and endonucleases. Prolonged stability was, in fact, found in model experiments with respective enzymes, as well as in studies done in human blood serum. Regardless of number and position of phosphoramidate linkages, the modified oligonucleotides showed only a slight decrease of Tm in hybridization studies with complementary oligonucleotides.