title: Targeted cross-linking of the human beta-globin gene in living cells mediated by a triple helix forming oligonucleotide.
abstract: 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.
authors.affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Kazi Abdus
authors.initials: KA
authors.lastname: Shahid
authors.forename: Alokes
authors.initials: A
authors.lastname: Majumdar
authors.forename: Rowshon
authors.initials: R
authors.lastname: Alam
authors.forename: Su-Ting
authors.initials: ST
authors.lastname: Liu
authors.forename: Jean Y
authors.initials: JY
authors.lastname: Kuan
authors.forename: Xuifen
authors.initials: X
authors.lastname: Sui
authors.forename: Bernard
authors.initials: B
authors.lastname: Cuenoud
authors.forename: Peter M
authors.initials: PM
authors.lastname: Glazer
authors.forename: Paul S
authors.initials: PS
authors.lastname: Miller
authors.forename: Michael M
authors.initials: MM
authors.lastname: Seidman
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000138:chemical synthesis; D000755:Anemia, Sickle Cell; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003562:Cytidine / Q000138:chemical synthesis; D003596:Cytosine / Q000031:analogs & derivatives; D004247:DNA / Q000494:pharmacology*; D005363:Ficusin / Q000494:pharmacology; D018390:Gene Targeting / Q000379:methods*; D005914:Globins / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020014:K562 Cells; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D010710:Phosphates / Q000737:chemistry; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D013696:Temperature; D014407:Tumor Cells, Cultured
chemical_list: D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D010710:Phosphates; D011687:Purines; D011743:Pyrimidines; C051802:triplex DNA; D003562:Cytidine; D003596:Cytosine; D005914:Globins; D004247:DNA; D000225:Adenine; D005363:Ficusin; C012518:pseudoisocytidine