Triplexes with 8-Aza-2′-Deoxyisoguanosine Replacing Protonated dC: Probing Third Strand Stability with a Fluorescent Nucleobase Targeting Duplex DNA

The fluorescent 8-aza-2′-deoxyisoguanosine (4) as well as the parent 2′-deoxyisoguanosine (1) were used as protonated dCH⁺ surrogates in the third strand of oligonucleotide triplexes. Stable triplexes were formed by Hoogsteen base pairing. In contrast to dC, triplexes containing nucleoside 1 or 4 in place of dCH⁺ are already formed under neutral conditions or even at alkaline pH values. Triplex melting can be monitored separately from duplex dissociation in cases in which the third strand contains the fluorescent nucleoside 4. Third-strand binding of oligonucleotides with 4, opposite to dG, was selective as demonstrated by hybridisation experiments studying mismatch discrimination. Third-strand binding is more efficient when the stability of the DNA duplex is reduced by mismatches, giving third-strand binding more flexibility.