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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 Article · · Nucleic Acids Research
DOI:https://doi.org/10.1093/nar/gkm664· OSTI ID:1625428
 [1];  [2];  [3];  [3];  [4];  [5];  [3];  [3];  [5];  [4];  [3];  [2]
  1. Vanderbilt Univ., Nashville, TN (United States). School of Medicine. Dept. of Biochemistry; DOE/OSTI
  2. Vanderbilt Univ., Nashville, TN (United States). School of Medicine. Dept. of Biochemistry
  3. Alnylam Pharmaceuticals, Inc., Cambridge, MA (United States). Dept. of Drug Discovery
  4. Northeastern Univ., Boston, MA (United States). Dept. of Chemistry and Chemical Biology
  5. Leopold-Franzens Univ., Innsbruck (Austria). Center for Molecular Biosciences (CMBI). Inst. of Organic Chemistry
+ Show Author Affiliations
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 ribo2,4-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 (TM) 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.
Research Organization:
Vanderbilt Univ., Nashville, TN (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
Grant/Contract Number:
W-31109-ENG-38
OSTI ID:
1625428
Journal Information:
Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 19 Vol. 35; ISSN 0305-1048
Publisher:
Oxford University PressCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (10)

Hydration Changes upon DNA Folding Studied by Osmotic Stress Experiments journal June 2012
High-Resolution NMR Analysis of the Conformations of Native and Base Analog Substituted Retroviral and LTR-Retrotransposon PPT Primers journal March 2008
2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling journal January 2012
Unexpected origins of the enhanced pairing affinity of 2′-fluoro-modified RNA journal December 2010
Exploring Chemical Modifications for siRNA Therapeutics: A Structural and Functional Outlook journal February 2010
On Stacking book November 2009
Synthesis, physicochemical and biological properties of oligonucleotides incorporated with amino-isonucleosides journal December 2011
Carbon-Oxygen Hydrogen Bonding in Biological Structure and Function journal October 2012
Synthesis, physicochemical and biological properties of oligonucleotides incorporated with amino-isonucleosides journal February 2012
Delivery of RNAi therapeutics: work in progress journal November 2013

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