H. Seliger

Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined. Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dim...

Oligonucleotide Conjugates Designed for Discriminative Hybridization at Physiological Temperature

Abstract We report the synthesis of oligonucleotide conjugates engineered to allow discriminative hybridization at temperatures around physiological. Two types of structural modifications were introduced: 1) internal oligomethylene and oligoethylene glycol spacers, and 2) terminal phenazinium residues. The thermal denaturation behaviour of the complexes formed by these oligonucleotide conjugates with a target sequence is compared to that of natural duplexes. We observed a lowering of the Tm of the duplexes formed by the internal modified oligonucleotides, whilst the terminal phenazinium residues enhance their stability. The effect of the spacers is modulated by their length and hydrophobic or hydrophilic nature. Alkylating substituents, which modify the target DNA strand on hybridization, were introduced on all conjugates, and the target cleavage obtained after piperidine treatment used as a further indicator of hybridization.

A Rapid Method for the Preparation of a One Dimensional Sequence-Overlapping Oligonucleotide Library

Abstract A new synthesis modul was constructed to prepare one-dimensional libraries of sequentally overlapping oligonucleotides on surface modified polypropylene tapes. The use of such a library for the diagnostic screening of a point mutation in the regulatory protein gene p53 is described.

SYNTHESIS AND ENZYMATIC STUDIES OF MODIFIED OLIGONUCLEOTIDES WITH ABIOLOGICAL MONOMER UNITS

Abstract The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block1 and the modified PNA-spacer (acyclic serinol nucleoside)2 can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.

Cleavage of RNA in hybrid duplexes by theE. coli ribonuclease H: II. Substrate properties of oligonucleotides containing nonnucleotide linkers

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for theE. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2–1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3′-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3′-3′-phosphodiester bond at the 3′-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity.

The support-on-support concept for in-situ oligonucleotide synthesis on nanoparticles.

Oligonucleotide-loaded nanoparticles, which are of interest for biomedical application, up to now, could not be prepared by in-situ synthesis, due to difficulty of handling in automated synthesizers. To overcome this problem, we have introduced the “support-on-support” concept. It is based on the reversible anchoring of nanoparticles to the surface of microparticles. These composite beads easily can be used for automated synthesis, being released after completion of chain elongations. As examples, dextran-coated magnetite nanoparticles were attached to polystyrene microparticles through (1) a gelatine or (2) a silica layer. Release involved dissolution of the bonding layer by (1) proteases or (2) alkali.

New Methods for the Solid-Phase Sequence Analysis of Nucleic Acid Fragments Using the Sanger Dideoxy Procedure

Abstract 3′-Terminal tailing of a given DNA fragment with 5-bromodeoxyuridine-5 triphosphate or biotinylated deoxyuridine-5′-triphosphate and deoxynucleotidyl terminal transferase allows its immobilization to an anti-bromo-deoxyuridine-antibody column or to a streptavidin column. The immobilized DNA could be subjected to enzymatic sequencing following the usual protocol. The dideoxy-nucleotide-terminated fragments were eluted with buffer containing formamide / dye mixture and directly applied to gel electrophoresis, allowing reading of ca. 600 bases. Several “sequencing cycles” could be performed with the same DNA column. Semi-mechanization of the process is described.

A VERSATILE POLYMER SUPPORT USEFUL FOR LARGE-SCALE SYNTHESIS OF DNA FOR BIOMEDICAL APPLICATIONS

Abstract A suitably derivatized MercKogelR 1 as an efficient solid support for the large-scale synthesis of DNA for biomedical applications, is described. Partial hydrolysis of these polyvinylacetate resins with 0.5 M NaOH solution1 at room temperature for 30–80 min. yielded carriers 1 that could be loaded with 120, 150, 258, and 368 μmoles of nucleosides g−1. At present a DAAD fellow in the University of Ulm, Section of Polymers, Germany.

An Automated Method to Create a One Dimensional Array of Chemically Synthesized Oligonucleotides

In this paper we describe a method to synthesize a variety of different oligonucleotides in a quasi parallel procedure on the surface of a polypropylene tape. The relation between necessary chemical coupling steps n and thus generated oligonucleotides m with a length of e.g. 15 monomers is: m = n − 14. To get a high synthesis throughput and the possibility of an overnight run, the system has been fully automated. To start the synthesis, only the desired sequence programming is necessary without further control of the system.