Design and synthesis of RNA miniduplexes via a synthetic linker approach.
Double-stranded oligodeoxyribonucleotides or single-stranded oligoribonucleotides with specific secondary structure have been proposed as potential antagonists to target nucleic acid-binding proteins (the sense approach). A major limitation of this strategy is that these derivatives are generally considered to be too large for pharmaceutical applications. We have developed a synthetic linker approach whereby nucleic acid duplexes of a much smaller size (miniduplexes) can be generated directly from a standard oligonucleotide synthesis. In this approach, four synthetic linkers (derivatized respectively from 1,9-nonanediol, triethylene glycol, 1,3-propanediol, and hexaethylene glycol) of different length and hydrophobicity were designed and incorporated into a model RNA molecule based on the TAR stem-loop structure of HIV-1. Their thermal stabilities were evaluated by measuring denaturation profiles (Tm measurements). These linker-derivatized RNA molecules were then assessed for their ability to bind to either a full-length protein (HIV-1 Tat protein) or a short peptide (Tat-derived peptide) through RNA mobility shift assays. Results from this study indicate that such modified miniduplex structures retain full binding activity relative to that of the wild-type sequence (Kd values), while Tm values were increased by 24-31 degrees C compared to an open duplex of the same length. This system provides a new direction in the use of nucleic acid miniduplexes as a novel class of oligonucleotide analogues for both fundamental research and possible therapeutic applications.
Ma
M Y
MY
Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.
Reid
L S
LS
Climie
S C
SC
Lin
W C
WC
Kuperman
R
R
Sumner-Smith
M
M
Barnett
R W
RW
D000595:Amino Acid Sequence; D001483:Base Sequence; D003198:Computer Simulation; D005026:Ethylene Glycols / Q000737:chemistry; D015696:Gene Products, tat / Q000378:metabolism; D006018:Glycols; D015497:HIV-1 / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D010446:Peptide Fragments / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011409:Propylene Glycols / Q000737:chemistry; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry; D054322:tat Gene Products, Human Immunodeficiency Virus
D005026:Ethylene Glycols; D015696:Gene Products, tat; D006018:Glycols; D010446:Peptide Fragments; D011409:Propylene Glycols; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; C077067:hexaethylene glycol; C028914:triethylene glycol; D011092:Polyethylene Glycols; C041787:1,3-propanediol; D012313:RNA; C079909:1,9-nonanediol