Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair.
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.
Broitman
S
S
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Amosova
O
O
Dolinnaya
N G
NG
Fresco
J R
JR
D000225:Adenine; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D011564:Furocoumarins; D005914:Globins / Q000235:genetics*; D006451:Hemoglobin, Sickle / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D017354:Point Mutation; D013698:Templates, Genetic; D013941:Thymine
D011564:Furocoumarins; D006451:Hemoglobin, Sickle; D009838:Oligodeoxyribonucleotides; D005914:Globins; D000225:Adenine; D013941:Thymine