Triplex crosslinking through furan oxidation requires perturbation of the structured triple-helix.

Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan-based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan-oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan-containing probes for the selective detection of single-stranded regions within nucleic acids containing a variety of structural motifs.