Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence

Abstract

Three identical deoxyoligonucleotide third strands with a 3'-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell beta-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target.

Figure 1

The deoxyoligonucleotide sequence of the β-globin gene fragment that includes the Sickle Cell mutation in codon 6 (a) or wild-type sequence (b). The mutant T residue and corresponding wild-type A residue are highlighted in bold. The gene target segment inserted into the plasmid is shown in (a). Note the duplex-forming hook at the 5′ end of the third strand, the combination of two third strand-binding motifs that enable strand switching, and the 5-methyl C (mC) and 5-propynyl U (pU) residues in the third strand. The psoralen moiety (Ps) is shown intercalated at the triplex–duplex junction.

Figure 2

Psoralen–third strand conjugates with varied linker lengths, N.

Figure 3

Kinetics of third strand-mediated psoralen photoproduct formation upon UVA irradiation. (a) Native PAGE of irradiated triplex mixtures with PsT-16 third strand. Similar gel patterns were obtained with PsT-6 and PsT-4. (b) Dependence of covalent triplex formation on UVA irradiation for each third strand. (c) PAGE of unirradiated triplexes with increasing PsT-6 third strand concentration in the standard buffer.

Figure 4

PAGE analysis of photoproducts formed with either target duplex strand and each third strand after irradiation with either 419 (PsT-4) or 365 nm light (Pst-4, PsT-6 and PsT-16).

Figure 5

Primer extension analysis of individual photoproducts formed with the D-1 duplex strand. (a) The major monoadduct is identified as T₁₁, and (b) the minor photoproduct as T₉ (see Fig. 1b).

Figure 6

Identification by primer extension arrest of an XL and of monoadducts at T₂₂, C₂₃, C₂₄, C₂₆, each formed with PsT-16 third strand. Lane D-2 shows the ladder obtained with the unmodified D-2 (non-coding) strand.

Figure 7

(a) Photoproducts formed with the wild-type duplex target sequence DH-1·DH-2 after UVA irradiation and (b) their identification by primer extension analysis as monoadducts at C₂₃, C₂₄, T₂₅. Lane DH-2 shows the ladder obtained with the unmodified DH-2 (non-coding strand).

Figure 8

Third strand binding to a plasmid containing the Sickle Cell β-globin target. Covalent attachment of the third strand reduces the mobility of the 299 bp restriction fragment containing the mutation site.