Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo.
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.
Alam
Md Rowshon
MR
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Majumdar
Alokes
A
Thazhathveetil
Arun Kalliat
AK
Liu
Su-Ting
ST
Liu
Ji-Lan
JL
Puri
Nitin
N
Cuenoud
Bernard
B
Sasaki
Shigeki
S
Miller
Paul S
PS
Seidman
Michael M
MM
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
D001952:Bridged-Ring Compounds; D002241:Carbohydrates; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; C051802:triplex DNA; D012266:Ribose; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D005363:Ficusin