Targeted Cross-linking of the Human β-Globin Gene in Living Cells Mediated by a Triple Helix Forming Oligonucleotide
| Autor: | Alokes Majumdar, Su Ting Liu, Peter M. Glazer, Rowshon Alam, Bernard Cuenoud, Jean Y. Kuan, Paul S. Miller, Kazi Abdus Shahid, Xuifen Sui, Michael M. Seidman |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Pyrimidine
Oligonucleotides Anemia Sickle Cell Cytidine Biology Biochemistry Phosphates Cytosine chemistry.chemical_compound Tumor Cells Cultured Humans Gene Base Sequence Oligonucleotide Adenine Ficusin Temperature Gene targeting DNA Hydrogen-Ion Concentration Molecular biology Globins A-site Cross-Linking Reagents Pyrimidines chemistry Purines Duplex (building) Gene Targeting Nucleic Acid Conformation K562 Cells Triple helix |
| Zdroj: | Biochemistry. 45:1970-1978 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi0520986 |
| Popis: | Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A8) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A8 did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m) and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|