Hybridization

Fig. 4.2.5. Homogeneous DNA detection with DNA-intercalator dye conjugates.

jugates 7 was up to 16 times higher than single-strand fluorescence. In one instance a hybridization-induced fluorescence decrease was observed. Similar fluorescence enhancements were determined on binding to single mismatched DNA as long as measurements were performed at temperatures below the melting temperature of the corresponding PNA-DNA duplex. DNA targets and their respective single-base mutants were distinguishable at temperatures that led to dissociation of mismatched complexes.

More selective detection of single-base mutations can be achieved by replacing a nucleobase with a fluorophore (Figure 4.2.6). We envisaged that this forced intercalation of fluorophores or fluorescent base surrogates should enable the detection hybridization rnprn

Fig. 4.2.6. A fluorophore that serves as fluorescent base surrogate is forced to intercalate adjacent to the expected mutation site. This forced intercalation enables detection of mismatched base pairs even within a formed duplex.

of structural changes caused by single base mutations or other DNA-modifying events [21]. For example, thiazole orange (TO) was attached to the backbone of the DNA-analogous peptide nucleic acid (Scheme 4.2.1). The TO was equipped with carboxyalkyl spacers of different length to enable coupling with the PNA backbone. To facilitate the screening of suitable TO derivatives we developed an on-resin procedure for gaining divergent access to a variety of PNA-dye conjugates which relied on the use of the internal modifier in 8. Combinatorial approaches uncovered the TO derivative in 9 as suitable fluorophore and revealed its preferred sequence context.

Scheme 4.2.1. Divergent solid-phase synthesis of FIT-probes.

For example, the thiazole orange-containing PNA-probe 10 was hybridized with oligonucleotides 11A and 11C (Figure 4.2.7) [O. Kohler, O. Seitz, Chem. Commun. 2003, DOI: 10.1039/B308299G]. Remarkably, the hybridization of 10 with 11A led to a 19-fold fluorescence increase. The fluorescence intensity of the single mismatched duplex 1711C was considerably lower (by a factor of 4) than the emission of matched duplex 1711C. These data suggest that FIT probes should become of high utility in diagnostic assays particularly in single-base-mutation analysis.

0 0

Post a comment