Dark times: iminothioindoxyl- C -nucleoside fluorescence quenchers with defined location and minimal perturbation in DNA.

Fluorescence quenchers for application in DNA - like the BHQ family - tend to be large molecules which need to be attached, often post-synthetically, via long linkers. In this study, we present two new iminothioindoxyl- C -nucleosidic quenchers which are very compact, feature a native backbone and can be introduced into DNA via regular solid-phase synthesis. Especially with d T as juxtaposed nucleobase, they have a defined location and orientation in a DNA duplex with minimal perturbation of the structure and hence interaction capabilities. Depending on the nature of the fluorophore, they can be used for orientation-(un)specific FRET studies. Their Förster radius is smaller than the one of BHQ-2. This makes these quenchers ideal for sophisticated studies using conditional quenching in the range between 470 and 670 nm in DNA.