PMID- 17881374
OWN - NLM
STAT- MEDLINE
DCOM- 20071227
LR  - 20190816
IS  - 1362-4962 (Electronic)
IS  - 0305-1048 (Print)
IS  - 0305-1048 (Linking)
VI  - 35
IP  - 19
DP  - 2007
TI  - 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.
PG  - 6424-38
AB  - 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.
FAU - Li, Feng
AU  - Li F
AD  - Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, 
      TN 37232, USA.
FAU - Pallan, Pradeep S
AU  - Pallan PS
FAU - Maier, Martin A
AU  - Maier MA
FAU - Rajeev, Kallanthottathil G
AU  - Rajeev KG
FAU - Mathieu, Steven L
AU  - Mathieu SL
FAU - Kreutz, Christoph
AU  - Kreutz C
FAU - Fan, Yupeng
AU  - Fan Y
FAU - Sanghvi, Jayodita
AU  - Sanghvi J
FAU - Micura, Ronald
AU  - Micura R
FAU - Rozners, Eriks
AU  - Rozners E
FAU - Manoharan, Muthiah
AU  - Manoharan M
FAU - Egli, Martin
AU  - Egli M
LA  - eng
GR  - R01 GM055237/GM/NIGMS NIH HHS/United States
GR  - R01 GM071461/GM/NIGMS NIH HHS/United States
GR  - R01 GM55237/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20070918
PL  - England
TA  - Nucleic Acids Res
JT  - Nucleic acids research
JID - 0411011
RN  - 0 (AGO2 protein, human)
RN  - 0 (Argonaute Proteins)
RN  - 0 (Eukaryotic Initiation Factor-2)
RN  - 0 (Fluorobenzenes)
RN  - 0 (Nucleotides)
RN  - 0 (Oligoribonucleotides)
RN  - 0 (RNA, Small Interfering)
RN  - 0 (RNA-Induced Silencing Complex)
RN  - 0 (ribo-2,4-difluorotoluylnucleotide)
SB  - IM
MH  - Argonaute Proteins
MH  - Base Pair Mismatch
MH  - Base Pairing
MH  - Crystallography, X-Ray
MH  - Eukaryotic Initiation Factor-2/*metabolism
MH  - Fluorobenzenes/*chemistry
MH  - HeLa Cells
MH  - Humans
MH  - Hydrogen Bonding
MH  - Models, Molecular
MH  - Nucleotides/*chemistry
MH  - Oligoribonucleotides/*chemistry/pharmacology
MH  - Osmotic Pressure
MH  - *RNA Interference
MH  - RNA, Small Interfering/*chemistry/pharmacology
MH  - RNA-Induced Silencing Complex/metabolism
MH  - Substrate Specificity
MH  - Thermodynamics
PMC - PMC2095806
EDAT- 2007/09/21 09:00
MHDA- 2007/12/28 09:00
PMCR- 2007/09/18
CRDT- 2007/09/21 09:00
PHST- 2007/09/21 09:00 [pubmed]
PHST- 2007/12/28 09:00 [medline]
PHST- 2007/09/21 09:00 [entrez]
PHST- 2007/09/18 00:00 [pmc-release]
AID - gkm664 [pii]
AID - 10.1093/nar/gkm664 [doi]
PST - ppublish
SO  - Nucleic Acids Res. 2007;35(19):6424-38. doi: 10.1093/nar/gkm664. Epub 2007 Sep 
      18.