Extensive Sugar Modification Improves Triple Helix Forming Oligonucleotide Activity in Vitro but Reduces Activity in Vivo
| Autor: | Arun K. Thazhathveetil, Ji Lan Liu, Su Ting Liu, Paul S. Miller, Bernard Cuenoud, Alokes Majumdar, Michael M. Seidman, Shigeki Sasaki, Nitin Puri, Rowshon Alam |
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| Rok vydání: | 2007 |
| Předmět: |
Bridged-Ring Compounds
Hypoxanthine Phosphoribosyltransferase Stereochemistry Ribose Kinetics Carbohydrates Oligonucleotides Nucleic Acid Denaturation Sensitivity and Specificity Biochemistry chemistry.chemical_compound Cricetinae Animals Thermal stability Base Pairing Binding Sites Oligonucleotide Ficusin Nucleic Acid Heteroduplexes Temperature Gene targeting DNA In vitro Electroporation Models Chemical chemistry Duplex (building) Gene Targeting Nucleic Acid Conformation Biological Assay Triple helix |
| Zdroj: | Biochemistry. 46:10222-10233 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi7003153 |
| Popis: | 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 T m 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. |
| Databáze: | OpenAIRE |
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