Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme.

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. Research Abstract Details 

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Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. Abstract Text:

Feng Li,Pradeep S Pallan,Martin A Maier,Kallanthottathil G Rajeev,Steven L Mathieu,Christoph Kreutz,Yupeng Fan,Jayodita Sanghvi,Ronald Micura,Eriks Rozners,Muthiah Manoharan,Martin Egli,Feng Li,Pradeep S Pallan,Martin A Maier,Kallanthottathil G Rajeev,Steven L Mathieu,Christoph Kreutz,Yupeng Fan,Jayodita Sanghvi,Ronald Micura,Eriks Rozners,Muthiah Manoharan,Martin Egli,

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA-protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176-183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 ('slicer' enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson-Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (T(M)) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. Publishing Authors By Initials

F Li,PS Pallan,MA Maier,KG Rajeev,SL Mathieu,C Kreutz,Y Fan,J Sanghvi,R Micura,E Rozners,M Manoharan,M Egli,F Li,PS Pallan,MA Maier,KG Rajeev,SL Mathieu,C Kreutz,Y Fan,J Sanghvi,R Micura,E Rozners,M Manoharan,M Egli,

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Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. Journal Published:

PUBLICATION TYPE: Research Support, U.S. Gov't,

Journal: Nucleic acids research

VOLUME: 35

Page Numbers: 6424-38

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ISSN: 1362-4962

DAY: 18

MONTH: 09

YEAR: 2007

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. Information

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LANGUAGE: eng

NlmUniqueID: 411011

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme. Keywords Mesh Terms:

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Grant and Affiliation Information for Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme.

AFFILIATION: Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.

Country: England

AGENCY: United States NIGMS

GRANT: R01 GM55237

ACRONYM: GM

MEDLINETA: Nucleic Acids Res

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