Thienoguanosine, a unique non-perturbing reporter for investigating rotational dynamics of DNA duplexes and their complexes with proteins.

Time-resolved fluorescence anisotropy (TRFA) provides key information on the dynamics of biomolecules and their interaction with ligands. However, since natural nucleosides are almost non-fluorescent, its application to DNA duplexes (dsDNA) requires fluorescent labels, which can alter dsDNA stability, hinder protein binding, and complicate interpretation of TRFA experiments due to their local motion. As shown here, thienoguanosine ( th G), a fluorescent analogue of guanosine, overcomes all these limitations. We recorded the TRFA decays of th G -labelled dsDNA of different lengths. th G behaved as a rigid, non-perturbing reporter, since no fast correlation time was recorded for any tested dsDNA. Due to its perfect stacking, only two correlation times, instead of the typical three, describe th G -labelled dsDNA rotational dynamics. Thanks to these features, we provided a complete description of the tumbling of the different dsDNA and their complexes with the Set and Ring Associated (SRA) domain of UHRF1, a key epigenetic regulator, obtaining values in excellent agreement with theoretical predictions. Moreover, th G was also found sensitive to SRA-induced base flipping of neighboring nucleobases. In the DNA label toolbox, th G thus stands out as a unique reporter for investigating the rotational dynamics of dsDNA and protein/dsDNA complexes. Thienoguanosine, a fluorescent analogue of guanosine, stands out in the DNA probe toolkit, as a rigid, non-perturbing reporter, with only two correlation times to describe dsDNA rotational dynamics. It allows a comprehensive description of the tumbling of dsDNA and their complexes with proteins, fully matching theoretical predictions. [Display omitted] • Thienoguanosine ( th G) behaves as a rigid, non-perturbing reporter in dsDNA. • Time-resolved fluorescence anisotropy of th G allows full dsDNA tumbling description. • Rotational correlation times of thG-labelled dsDNA ± protein fully match with theory • th G is sensitive to protein-induced base flipping of neighboring nucleobases • th G is unique in the toolkit of fluorescent DNA labels for monitoring DNA dynamics. [ABSTRACT FROM AUTHOR]