Alex Andrus

AN EFFICIENT METHOD FOR THE ISOLATION AND PURIFICATION OF OLIGORIBONUCLEOTIDES

Abstract Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.

Fast oligonucleotide deprotection phosphoramidite chemistry for DNA synthesis

Abstract A new set of base protecting groups for cyanoethylphosphoramidite nucleosides and supports has been developed which decreases the post-synthesis time requirements. The traditional purine amide protecting groups, which require 8-16 hours at 55°C for deprotection in ammonia, have been replaced with the dimethylformamidine group. Oligonucleotides made with the new reagents require only 1 hour at 55°C or 8 hours at room temperature for complete deprotection. Dialkylformamidine phosphoramidites exhibit enhanced resistance to depurination compared to the traditional, or even the phenoxyacetyl, phosphoramidites.

Large scale synthesis of oligonucleotides via phosphoramidite nucleosides and a high-loaded polystyrene support

Abstract Large scale quantities of phosphodiester and phosphorothioate oligonucleotides are synthesized on an aminopolyethyleneglycol derivatized polystyrene (TentaGel) support. Efficient, automated synthesis up to 1 mmole scale is achieved with phosphoramidite nucleoside monomers and 5-ethylthiotetrazole activator.

An optimized polystyrene support for rapid, efficient oligonucleotide synthesis

Abstract An optimized type of polystyrene has been developed as a solid support for automated oligonucleotide synthesis. The non-swelling, rigid beads possess the attractive features of rapid reaction kinetics, efficient washing with organic solvents, and mechanical stability. Pore size, particle size, and polymerization formula and conditions have been optimized. The support material is derivatized to give primary amino functionality, which is near quantitatively coupled to 3′ p-nitrophenyl succinate nucleosides. Loading of the nucleosides can be precisely controlled in a range of 5 to 70 μmole per gram. The high yield synthesis of oligonucleotides, by phosphoramidite chemistry has been demonstrated up to 120 bases in length. Side reactions, such as extraneous chain growth, are minimized due to the lack of reactive functionality on the new support surface.

Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis

Abstract Improvement of the hydrogen-phosphonate method for oligodeoxynucleotide synthesis has been achieved via an efficient capping reagent, triethylammonium isopropylphosphite, resulting in stable 5′ isopropylphosphate failure sequences. Activation for capping and coupling is best effected with 1-adamantanecarbonyl chloride.

A convenient automated solid-phase synthesis of PNA-(5′)-DNA-(3′)-PNA chimera

An automated online solid-phase synthesis of Ac-cac ct T∗GG TC t∗ac ct-Gly-OH1 using standard DNA and appropriately protected PNA building blocks (2–5) is described. This chimera forms stable duplexes with complementary DNA and RNA.

Automated synthesis of double dye-labeled oligonucleotides using tetramethylrhodamine (TAMRA) solid supports

TAMRA fluorescent dye-labeled non-nucleosidic synthesis supports have been derivatized for automated synthesis of 3′ dye-labeled oligonucleotides.1 Synthesis with 5′ fluorescent dye phosphoramidites leads to 5′ and 3′ double labeled oligonucleotides, useful in Taqman assay and FRET experiments. Cleavage and deprotection of dye-labeled oligonucleotides are carried out with tert-butylamine:methanol:water (1:1:2) without any degradation and/or modification of dyes and nucleobases.

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology.

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

The synthesis of N-(tetrazol-5-yl)azetidin-2-ones

Abstract A series of N-(tetrazol-5-yl)azetidin-2-ones have been prepared from serine and threonine and evaluated as antibiotics against a range of bacteria.

N-6-Dialkylformamidine-2′-deoxyadenosine Phosphoramidites in Oligodeoxynucleotide Synthesis Raped Deprotection of Oligodeoxynucleotides

Abstract A series of dialkylformamidine protected deoxyadenosine phosphoramidites were prepared for automated, solid-support DNA synthesis. The set of Abz, Gdmf, Cbz, T phosphoramidites gave high purity and high yield oligodeoxynucleotides, with complete deprotection at 65 °C in one hour. Different times and temperatures of exposure to concentrated ammonium hydroxide were examined to establish the optimum conditions for deprotection of oligodeoxynucleotides.