Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair

• Published in: The Journal of biological chemistry 1999-07, Vol.274 (31), p.21763-21768
• Main Authors: Broitman, S, Amosova, O, Dolinnaya, N G, Fresco, J R
• Format: Article
• Language: English
• Subjects: Adenine; Base Pairing; Base Sequence; Binding Sites; DNA Footprinting; Furocoumarins; Globins - genetics; Hemoglobin, Sickle - genetics; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemical synthesis; Oligodeoxyribonucleotides - chemistry; Point Mutation; Templates, Genetic; Thymine
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.31.21763

A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.