Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing.
We report that combining a DNA analog (2'F-ANA) with rigid RNA analogs [2'F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1-1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.
Deleavey
Glen F
GF
Department of Chemistry, McGill University, Montréal, QC, Canada.
Watts
Jonathan K
JK
Alain
Tommy
T
Robert
Francis
F
Kalota
Anna
A
Aishwarya
Veenu
V
Pelletier
Jerry
J
Gewirtz
Alan M
AM
Sonenberg
Nahum
N
Damha
Masad J
MJ
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D001088:Arabinonucleotides / Q000737:chemistry*; D018797:Cell Cycle Proteins; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000096:biosynthesis; D049409:Luciferases, Firefly / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry*; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism
C507672:2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid; D048868:Adaptor Proteins, Signal Transducing; D001088:Arabinonucleotides; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; C104151:EIF4EBP2 protein, human; D039642:Eukaryotic Initiation Factors; D009841:Oligonucleotides; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C477371:locked nucleic acid; D012313:RNA; D004247:DNA; D007372:Interferons; D049409:Luciferases, Firefly