Minimum Number of 2‘-O-(2-Aminoethyl) Residues Required for Gene Knockout Activity by Triple Helix Forming Oligonucleotides
| Autor: | Nitin Puri, Pierre Martin, Paul S. Miller, Alokes Majumdar, Michael M. Seidman, Bernard Cuenoud, Andre Boyd, Francois Natt |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Hypoxanthine Phosphoribosyltransferase
Hot Temperature Pyrimidine Ribose Oligonucleotides CHO Cells Biology Biochemistry Cytosine chemistry.chemical_compound Cricetulus Cricetinae Animals Gene knockout Oligonucleotide DNA A-site Electroporation chemistry Duplex (building) Gene Targeting Nucleic Acid Conformation Thymidine Triple helix |
| Zdroj: | Biochemistry. 41:7716-7724 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi025734y |
| Popis: | Triple helix forming oligonucleotides (TFOs) that bind chromosomal targets in living cells may become tools for genome manipulation, including gene knockout, conversion, or recombination. However, triplex formation by DNA third strands, particularly those in the pyrimidine motif, requires nonphysiological pH and Mg(2+) concentration, and this limits their development as gene-targeting reagents. Recent advances in oligonucleotide chemistry promise to solve these problems. For this study TFOs containing 2'-O-methoxy (2'-OMe) and 2'-O-(2-aminoethyl) (2'-AE) ribose substitutions in varying proportion have been constructed. The TFOs were linked to psoralen and designed to target and mutagenize a site in the hamster HPRT gene. T(m) analyses showed that triplexes formed by these TFOs were more stable than the underlying duplex, regardless of 2'-OMe/2'-AE ratio. However, TFOs with 2'-AE residues were more stable in physiological pH than those with only 2'-OMe sugars, as a simple function of 2'-AE content. In contrast, gene knockout assays revealed a threshold requirement--TFOs with three or four 2'-AE residues were at least 10-fold more active than the TFO with two 2'-AE residues. The HPRT knockout frequencies with the most active TFOs were 300-400-fold above the background, whereas there was no activity against the APRT gene, a monitor of nonspecific mutagenesis. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|