title: Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo.
abstract: We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.
authors: Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors: Md Rowshon
authors: MR
authors: Alam
authors: Alokes
authors: A
authors: Majumdar
authors: Arun Kalliat
authors: AK
authors: Thazhathveetil
authors: Su-Ting
authors: ST
authors: Liu
authors: Ji-Lan
authors: JL
authors: Liu
authors: Nitin
authors: N
authors: Puri
authors: Bernard
authors: B
authors: Cuenoud
authors: Shigeki
authors: S
authors: Sasaki
authors: Paul S
authors: PS
authors: Miller
authors: Michael M
authors: MM
authors: Seidman
mesh_terms: D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D001681:Biological Assay; D001952:Bridged-Ring Compounds / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D006224:Cricetinae; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D018274:Electroporation; D005363:Ficusin / Q000737:chemistry; D018390:Gene Targeting / Q000379:methods; D007041:Hypoxanthine Phosphoribosyltransferase; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides / Q000737:chemistry* / Q000494:pharmacology; D012266:Ribose / Q000031:analogs & derivatives / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D001952:Bridged-Ring Compounds; D002241:Carbohydrates; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; C051802:triplex DNA; D012266:Ribose; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D005363:Ficusin