Thiazole orange-carboplatin triplex-forming oligonucleotide (TFO) combination probes enhance targeted DNA crosslinking.

We report a new class of carboplatin-TFO hybrid that incorporates a bifunctional alkyne-amine nucleobase monomer called AP-C3-dT that enables dual 'click' platinum(ii) drug conjugation and thiazole orange fluorophore coupling. Thiazole orange enhances the binding of Pt(ii)-TFO hybrids and provides an intrinsic method for monitoring triplex formation. These hybrid constructs possess increased stabilisation and crosslinking properties in comparison to earlier Pt(ii)-TFOs, and demonstrate sequence-specific binding at neutral pH.

A new phosphoramidite enables orthogonal double labelling to form combination oligonucleotide probes.

Oligonucleotides labelled with thiazole orange intercalator and a reporter dye on the same thymine base have been synthesized. The key phosphoramidite (AP-C3 dT) contains an alkyne and amine, enabling dual orthogonal labelling of the nucleobase. Multiple monomers can be added to produce heavily functionalised oligonucleotides.

Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting.

We have synthesised a range of thiazole orange (TO) functionalised oligonucleotides for nucleic acid detection in which TO is attached to the nucleobase or sugar of thymidine. The properties of duplexes between TO-probes and their DNA and RNA targets strongly depend on the length of the linker between TO and the oligonucleotide, the position of attachment of TO to the nucleotide (major or minor groove) and the mode of attachment of thiazole orange (via benzothiazole or quinoline moiety). This information can be used to design probes for detection of target nucleic acids by fluorescence or duplex melting.