Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring
Vladimir B Tsvetkov1, Anton V Turaev2, Nataliia A Petrunina3
1Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, 119435, Russia and World-Class Research Center "Digital biodesign and personalized healthcare", Sechenov First Moscow State Medical University, 8/2 Trubetskaya Str., Moscow, 119146, Russia and A.V. Topchiev Institute of Petrochemical Synthesis RAS, Moscow, 119071, Russia.
Abstract:
The lack of high throughput screening (HTS) techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing the effects of ligands on the iM stability using the FAM-BHQ1 pair provides incorrect results due to additional dye-related interactions. We developed an alternative system with fluorescent phenoxazine pseudonucleotides in loops that do not alter iM unfolding. At the same time, the fluorescence of phenoxazine residues is sensitive to iM unfolding that enables accurate evaluation of ligand-induced changes of iM stability. Our results provide the basis for new approaches for HTS of iM ligands.
