Mechanism and Specificity of RNA Cleavage by Chemical Ribonucleases

Abstract

Cleaving of model RNA substrates by chemical ribonucleases constructed by conjugation of 1,4 diazabicyclo[2,2,2]octane with histamine and histidine was investigated. Similarly to RNase A, the chemical RNases produce fragments with 5′ hydroxy-group and 3′-cyclophosphate. The cleavage occurs as the catalytic reaction: more than 150 phosphodiester bonds in RNA can be cleaved by one molecule of RNase mimic.     

Simple and rapid procedure suitable for quantitative isolation of low and high molecular weight extracellular nucleic acids

The procedure based on binding of nucleic acids with glass surface in presence of chaotropic salts was adapted for efficient isolation of 100-10000 b.p. DNA fragments and 50-10,000 b. RNA fragments. The method provide 90% and 85% efficacy of isolation of 100 b.p. DNA and 100 b. RNA fragments respectively. High molecular weight nucleic acids are isolated with 98% efficacy. Isolated nucleic acids are free from contaminations, influencing nucleic acids modifying enzymes and fluorochromes. The method is rapid, simple and cost-effective.     

Extracellular nucleic acids

Extracellular nucleic acids are found in different biological fluids in the organism and in the environment: DNA is a ubiquitous component of the organic matter pool in the soil and in all marine and freshwater habitats. Data from recent studies strongly suggest that extracellular DNA and RNA play important biological roles in microbial communities and in higher organisms. DNA is an important component of bacterial biofilms and is involved in horizontal gene transfer. In recent years, the circulating extracellular nucleic acids were shown to be associated with some diseases. Attempts are being made to develop noninvasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Recent observations demonstrated the possibility of nucleic acids exchange between eukaryotic cells and extracellular space suggesting their participation in so far unidentified biological processes.     

Design and synthesis of metal-free artificial ribonucleases

The present review is aimed at giving a general overview of our results in the field of designing and synthesizing simple peptide-like molecules that mimic structural and functional aspects of natural ribonucleases, as well as designing oligonucleotide-based artificial ribonucleases.     

Mapping the amino acid properties of constituent nucleoporins onto the yeast nuclear pore complex

Visualization of molecular structures aids in the understanding of structural and functional roles of biological macromolecules. Macromolecular transport between the cell nucleus and cytoplasm is facilitated by the nuclear pore complex (NPC). The ring structure of the NPC is large and contains several distinct proteins (nucleoporins) which function as a selective gate for the passage of certain molecules into and out of the nucleus. In this note we demonstrate the utility of a python code that allows direct mapping of the physiochemical properties of the constituent nucleoporins on the scaffold of the yeast NPC׳s cytoplasmic view. We expect this tool to be useful for researchers to visualize the NPC based on their physiochemical properties and how it alters when specific mutations are introduced in one or more of the nucleoporins. The code developed using Python is available freely from the authors.     

Artificial ribonucleases: from combinatorial libraries to efficient catalysts of RNA cleavage

Combinatorial libraries of small organic compounds capable of cleaving RNA were synthesized. The compounds contain benzene ring substituted with two residues of bis quaternary salt of diazabicyclo[2.2.2]octane (DABCO) bearing hydrophobic fragments of different length and structure, attached to DABCO at the bridge position. These compounds, lacking traditional functionalities involved in transesterification reaction, exhibit pronounced RNA cleavage activity. To identify the most active artificial ribonucleases, sublibraries and truncated libraries, containing compounds lacking one of substituents were synthesized. Analysis of ribonuclease activity of truncated libraries resulted in identification of the most active compounds, which are characterized by the presence of at least one long oligomethylene substituent.     

Cleavage of RNA by an amphiphilic compound lacking traditional catalytic groups

Recently, in experiments with combinatorial libraries of amphiphilic compounds lacking groups, known as catalysts of transesterification reaction, we discovered novel RNA-cleaving compounds [N. Kovalev, E. Burakova, V. Silnikov, M. Zenkova, V. Vlassov, Bioorg. Chem. 34 (2006) 274-286]. In the present study, we investigate cleavage of RNA by the most active representative of these libraries, compound named Dp12. Sequence-specificity of RNA cleavage and influence of reaction conditions on cleavage rate suggested that Dp12 enormously accelerates spontaneous RNA cleavage. Light scattering experiments revealed that the RNA cleavage proceeds within multiplexes formed by assembles of RNA and Dp12 molecules, at Dp12 concentration far below critical concentration of micelle formation. Under these conditions, Dp12 is presented in the solution as individual molecules, but addition of RNA to this solution triggers formation of the multiplexes. The obtained data suggest a possible mechanism of RNA cleavage, which includes interaction of the compound with RNA sugar-phosphate backbone resulting in changing of ribose conformation. This leads to juxtaposition of the 2′-hydroxyl group and internucleotide phosphorus atom at a distance needed for the transesterification to occur.