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 tr ipler with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Tripler-mediated psoralen modification of th e mutant T residue was sought as an approach to gene repair. The 24-nucleot ide purine-rich target sequence switches from one strand to the other and h as four pyrimidine interruptions. Therefore, a third strand sequence favora ble to two tripler motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third stran d binding, 5-methylcytosine and 5-propynyluracil were used in the third str and. 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 tripler formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the thir d strand forms photoadducts to the targeted T with high efficiency. Such mo noadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.