A. V. Aralov

Synthesis Of RNA by the Rapid Phosphotriester Method Using Azido-Based 2′-O-Protecting Groups

The azidomethyl and 2-(azidomethyl)benzoyl as 2′-OH protecting groups are reported for preparation of oligoribonucleotides by the phosphotriester solid-phase method using O-nucleophilic intramolecular catalysis. The procedures for the synthesis of the corresponding monomer synthons were developed and the usefulness of the application of 2′-O-azidomethyl and 2′-O-2-(azidomethyl)benzoyl groups was examined in the synthesis of different RNA fragments with a chain length of 15–22 nucleotides. The azidomethyl group was found to be more preferable for effective synthesis of oligoribonucleotides. Hybridization properties of RNAs toward their complementary oligonucleotides were examined before and after the removal of 2′-O-azidomethyl groups.

An azidomethyl protective group in the synthesis of oligoribonucleotides by the phosphotriester method

A rapid and effective method of an automatic oligoribonucleotide synthesis alternative to the phosphoramidite one was developed. This method is based on the phosphotriester approach to internucleotide bond formation under intramolecular O-nucleophilic catalysis and the use of an azidomethyl group for protection of a nucleotide 2′-hydroxyl function.

DNA mimics based on pyrrolidine and hydroxyproline

In recent years, a great number of analogues and mimics of nucleic acids have been developed with the aim of improving the physicochemical and biological properties of native oligonucleotides, in particular, to increase their affinity for nucleic acids, selectivity of action, and biological stability. This review summarizes the data on the synthesis and properties of DNA mimics, the analogues of peptide nucleic acids, which are the derivatives of pyrrolidine and hydroxyproline. Some physicochemical and biological properties of negatively charged mimics of this type are considered, which contain phosphonate residues in the back-bone and exhibit a high affinity for DNA and RNA, the selectivity of binding to nucleic acids, and stability in various biological systems. Examples of using these mimics as tools in molecular biology studies, in particular, functional genomics, are given. The prospects for their application in diagnosis and medicine are discussed.

N-azidomethylbenzoyl blocking group in the phosphotriester synthesis of oligoribonucleotides

An effective modification of the phosphotriester method has been developed for the automatic synthesis of DNA and RNA fragments using O-nucleophilic intramolecular catalysis and the 2-(azidomethyl)benzoyl group for the protection of the amino groups of nucleotide heterocyclic bases.

i-Clamp phenoxazine for the fine tuning of DNA i-motif stability

Abstract Non-canonical DNA structures are widely used for regulation of gene expression, in DNA nanotechnology and for the development of new DNA-based sensors. I-motifs (iMs) are two intercalated parallel duplexes that are held together by hemiprotonated C-C base pairs. Previously, iMs were used as an accurate sensor for intracellular pH measurements. However, iM stability is moderate, which in turn limits its in vivo applications. Here, we report the rational design of a new substituted phenoxazine 2′-deoxynucleotide (i-clamp) for iM stabilization. This residue contains a C8-aminopropyl tether that interacts with the phosphate group within the neighboring chain without compromising base pairing. We studied the influence of i-clamp on pH-dependent stability for intra- and intermolecular iM structures and found the optimal positions for modification. Two i-clamps on opposite strands provide thermal stabilization up to 10–11°C at a pH of 5.8. Thus, we developed a new modification that shows significant iM-stabilizing effect both at strongly and mildly acidic pH and increases iM transition pH values. i-Clamp can be used for tuning iM-based pH probes or assembling extra stable iM structures for various applications.

Protective Groups in the Chemical Synthesis of Oligoribonucleotides

The methods of chemical oligoribonucleotide synthesis and the protective groups used are reviewed. The latest data on the protection of 2′-OH functions of nucleotide derivatives used as monomers for the RNA synthesis are comprehensively discussed.

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in synthesis of oligoribunucleotides by the phosphotriester method

An efficient method to synthesize monomer ribonucleotide synthons containing 2′-O-methoxymethyl and 2′-O-(p-nitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligonucleotide phosphotriester method using O-nucleophilic intramolecular catalysis at the stage of the internucleotide bond formation. The former synthons may be used for the automatic synthesis of 2′-modified oligonucleotides; the latter synthons made be used for the synthesis of phosphotriester oligoribonucleotides in high yields.

Monomers containing 2′-O-alkoxymethyl groups as synthons for the oligonucleotide synthesis by the phosphotriester method

A general scheme for the synthesis of ribonucleotides containing an alkoxymethyl group at the 2′-O-position of ribose and an O-nucleophilic catalytic 4-methoxy-1-oxido-2-picolyl phosphate-protecting group has been developed for the introduction into oligonucleotides during their solid-phase synthesis by the phosphotriester method. The scheme has been tested in the synthesis of monomers with 2′-O-modifying groups as examples: 2-azidoethoxymethyl, propargyloxymethyl, and 3,4-cyclocarbonatebutoxymethyl groups.

A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development

BackgroundThe initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone – the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis.ResultsFor a more detailed comparison of steroidogenic systems of Plantae and Animalia, we have created and studied transgenic tobacco and tomato plants efficiently expressing mammalian CYP11A1 cDNA. The detailed phenotypic characterization of plants obtained has shown that through four generations studied, the transgenic tobacco plants have reduced a period of vegetative development (early flowering and maturation of bolls), enlarged biomass and increased productivity (quantity and quality of seeds) as compared to the only empty-vector containing or wild type plants. Moreover, the CYP11A1 transgenic plants show resistance to such fungal pathogen as Botrytis cinerea. Similar valuable phenotypes (the accelerated course of ontogenesis and/or stress resistance) are also visible in two clearly distinct transgenic tomato lines expressing CYP11A1 cDNA: one line (No. 4) has an accelerated rate of vegetative development, while the other (No. 7) has enhanced immunity to abiotic and biotic stresses. The progesterone level in transgenic tobacco and tomato leaves is 3–5 times higher than in the control plants of the wild type.ConclusionsFor the first time, we could show the compatibility in vivo of even the most specific components of the systems of biosynthesis of steroid hormones in Plantae and Animalia. The hypothesis is proposed and substantiated that the formation of the above-noted special phenotypes of transgenic plants expressing mammalian CYP11A1 cDNA is due to the increased biosynthesis of progesterone that can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals.

Synthesis of Oligoribonucleotides Containing 2′-O-Methoxymethyl Group by the Phosphotriester Method

An effective procedure for the synthesis of ribonucleotide monomers containing a 2 ′-О-methoxymethyl-modifying group was developed. These monomers were used for the synthesis of RNA fragments by the solid-phase phosphotriester method under O-nucleophilic intramolecular catalysis. The properties of 2 ′-О-methoxymethyl-containing oligoribonucleotides were examined.