How does Hoechst bind to DNA?

Hoechst binds to the minor groove of double-stranded DNA. It shows a marked preference for binding with sequences rich in adenine and thymine. Hoechst 33258 is the most commonly used dye for DNA staining. It is used as a starting compound for the synthesis of more complex sensors or probes. Hoechst 33258 possesses a phenolic (-OH) group, which can be used for the attachment of the linker by standard Williamson ether synthesis conditions or can be readily transformed to triflate and further coupled to a moiety of interest by a transition metal catalysed cross-coupling reaction. It binds to DNA in two different modes: the high affinity (K_d 1–10 nM) binding from the specific interaction with B-DNA minor groove and the low affinity (K_d ~1000 nM) due to the nonspecific interaction with DNA sugar–phosphate backbone. The optimal binding site is AAA/TTT.