Vladimir A. Efimov

Complete nucleotide sequence and genome organization of a tobamovirus infecting cruciferae plants

Genomic RNA sequence of a tobamovirus infecting cruciferae plants (cr‐TMV) was determined. The RNA is composed of 6312 nucleotides and contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), 29K (ORF3) and 18K (capsid protein, ORF4). ORF4 overlaps ORF3 by 74 nucleotides and the overlapping region can be folded into a stable hairpin structure. The 3′‐terminal region of the cr‐TMV RNA preceding the tRNA‐like structure was shown to form six potentially stable pseudoknots.

An approach towards the synthesis of oligomers containing a N-2-hydroxyethyl-aminomethylphosphonate backbone: A novel PNA analogue

A convenient route to the preparation of 4-methoxy-1-oxido-pyridine-2-methyl N-2-(4,4′-dimethoxytrityloxy)ethyl-N-thymin-1-yl-aminomethylphosphonate (1a, T∗) and the corresponding N4-benzoylcytosin-1-yl derivative 1b (C∗) is reported. These PPNA monomers proved to be suitable building blocks in a solid-support synthesis of the tetradecameric fragment (C∗T∗T∗T∗C∗T∗T∗T∗T∗C∗T∗C∗T∗)dT.

PNA-RELATED OLIGONUCLEOTIDE MIMICS AND THEIR EVALUATION FOR NUCLEIC ACID HYBRIDIZATION STUDIES AND ANALYSIS

DNA mimics containing phosphonate analogues of PNAs (pPNAs), particularly PNA-pPNA hybrids as well as hetero-oligomers consisted of pPNA units and PNA-like molecules on the base of trans-4-hydroxy-L-proline (HypNA) have been synthesized. The evaluation of their effectiveness in assays based on the hybridization technique in the comparison with natural oligonucleotides and classical PNAs has shown a high potential of these mimics as sensor molecules for nucleic acid based diagnostics and as molecular probes for mRNA isolation.

Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms.

Synthesis and application of negatively charged PNA analogues.

Negatively charged DNA mimics containing phosphonate analogues of peptide nucleic acids were designed, and their physicochemical and biological properties were evaluated in the comparison with natural oligonucleotides, classical peptide nucleic acids, and morpholino phosphorodiamidate oligonucleotide analogues. The results obtained revealed a high potential of phosphonate-containing PNA derivatives for a number of biological applications, such as diagnostic, nucleic acids analysis, and inhibition of gene expression.

Phosphono Peptide Nucleic Acids with a Constrained Hydroxyproline-Based Backbone

Abstract DNA mimics representing negatively charged analogues of peptide nucleic acids (PNAs), particularly hetero-oligomers constructed from alternating phosphono-PNA residues (pPNA) and monomers on the base of trans-4-hydroxy-L-proline (HypNA) as well as mimics composed of phosphono-HypNA monomers (pHypNA) were tested in a set of in vitro and in vivo assays, and they demonstrated a high potential for the use in nucleic acid based diagnostic, isolation of nucleic acids and antisense experiments.

Approach to the Synthesis of Natural and Modified Oligonucleotides by the Phosphotriester Method Using O-Nucleophilic Intramolecular Catalysis

An approach to the solid phase synthesis of natural and modified oligonucleotides using phosphotriester technique has been developed. Particularly, this method allows the synthesis of ribo- and deoxyribo-oligonucleotides containing various 2 ′-modified mononucleotides as well as stereodefined nucleotide phosphorothioate analogues.

MESSENGER RNA ISOLATION USING NOVEL PNA ANALOGUES

Homo-Thy hetero-oligomer probes composed of trans-4-hydroxy-L-proline based PNA-like (HypNA) monomers and phosphono PNA (pPNA) monom-ers demonstrated strong binding to complementary poly A+ RNA strands. We used a mixture of chimeric oligomers containing both “linear” and “clamping” PNA-analogues to develop an mRNA isolation procedure and demonstrate the improved recovery of RNA molecules with secondary structure at the 3′end as well as RNAs with short poly A tails.

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.

Synthesis of DNA analogues with novel carboxamidomethyl phosphonamide and glycinamide internucleoside linkages.

Thymidine oligonucleotide analogues with phosphodiester bonds fully substituted by carboxamidomethyl phosphonamide, or glycinamide linkages were synthesized on a solid support, and their hybridization properties toward DNA and RNA targets were determined by Tm analysis.