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      Site-Selective Artificial Ribonucleases: Oligonucleotide Conjugates Containing Multiple Imidazole Residues in the Catalytic Domain

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          Abstract

          Design of site-selective artificial ribonucleases (aRNases) is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containing multiple imidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.

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          Most cited references79

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          Bridging the gap between proteins and nucleic acids: a metal-independent RNAseA mimic with two protein-like functionalities.

          Two synthetically modified nucleoside triphosphate analogues (adenosine modified with an imidazole and uridine modified with a cationic amine) are enzymatically polymerized in tandem along a degenerate DNA library for the combinatorial selection of an RNAse A mimic. The selected activity is consistent with both electrostatic and general acid/base catalysis at physiological pH in the absence of divalent metal cations. The simultaneous use of two modified nucleotides to enrich the catalytic repertoire of DNA-based catalysts has never before been demonstrated and evidence of general acid/base catalysis at pH 7.4 for a DNAzyme has never been previously observed in the absence of a divalent metal cation or added cofactor. This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.
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            Cleavage of RNA phosphodiester bonds by small molecular entities: a mechanistic insight.

            RNA molecules participate in many fundamental cellular processes either as a carrier of genetic information or as a catalyst, and hence, RNA has received increasing interest both as a chemotherapeutic agent and as a target of chemotherapy. In addition the dual nature of RNA has led to the RNA-world concept, i.e. an assumption that the evolution at an early stage of life was based on RNA-like oligomers that were responsible for the storage and transfer of information and as catalysts maintained primitive metabolism. Accordingly, the kinetics and mechanisms of the cleavage of RNA phosphodiester bonds have received interest and it is hoped they will shed light on the mechanisms of enzyme action and on the development of artificial enzymes. The major mechanistic findings concerning the cleavage by small molecules and ions and their significance for the development of efficient and biologically applicable artificial catalysts for RNA hydrolysis are surveyed in the present perspective.
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              The reactivity of phosphodiester bonds within linear single-stranded oligoribonucleotides is strongly dependent on the base sequence.

              The cleavage of short chimeric oligonucleotides containing only one reactive ribonucleoside unit, all other nucleosides being 2'-O-methylated, has been studied at pH 8.5 and 35 degrees C. Among the 20 different sequences that did not exhibit any tendency to form a defined secondary structure, the scissile 5'-UpA-3' and 5'-CpA-3' phosphodiester bonds experienced >100- and up to 35-fold reactivity differences, respectively. Compared with dinucleoside monophosphates, both rate accelerations and retardations of more than one order of magnitude were observed. Even a change of a single base several nucleosides away from the scissile bond markedly affected the reaction rate. Duplex formation at the 3'- and/or 5'-side of the scissile bond was also studied and observed to be strongly rate retarding. The origin of the high sensitivity of phosphodiester bonds to the molecular environment is discussed.
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                Author and article information

                Journal
                J Nucleic Acids
                JNA
                Journal of Nucleic Acids
                SAGE-Hindawi Access to Research
                2090-0201
                2090-021X
                2011
                25 September 2011
                : 2011
                : 748632
                Affiliations
                1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia
                2School of Pharmacy and Pharmaceutical Sciences, The University of Manchester, Manchester M13 9PL, UK
                3Fidelity Systems Inc., 7961 Cessna Avenue, Gaithersburg, MD 20879, USA
                Author notes
                *Marina A. Zenkova: marzen@ 123456niboch.nsc.ru

                Academic Editor: Dmitry A. Stetsenko

                Article
                10.4061/2011/748632
                3180074
                21961054
                8ae4b056-7038-45e5-9eca-6688f9fbd89c
                Copyright © 2011 Natalia G. Beloglazova et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 7 April 2011
                : 5 July 2011
                Categories
                Research Article

                Genetics
                Genetics

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