A fluorescent probe under the microscope showing dual recognition of B-DNA and G-quadruplex DNA

The use of small molecules as fluorescent DNA markers has been extensively explored since the mid-1960s, revealing limitations in designing selective probes due to the largely missing relationship between probe structure and DNA conformation. Here, we present a multistep computational protocol applied to a recently proposed fluorescent marker, QCy(MeBT)₃, capable of simultaneously recognizing both B-DNA and G-quadruplex DNA through distinct emission signatures. Our protocol identifies the conformations that enable selective probe–DNA binding and predicts a remarkably high affinity. Moreover, these same conformations are responsible for the different absorption and fluorescence shifts observed experimentally upon binding to B-DNA or G-quadruplex. Overall, we establish a fundamental principle for predicting the performance of fluorescent probes in complex biological environments: the properties of specific molecular conformations determine their photobiophysical fate upon binding to a given DNA sequence, and thus their ability to recognize a particular DNA topology. Although the use of small molecules as fluorescent markers for detecting different DNA conformations has been explored extensively, achieving specific structural relationships between probe and DNA conformations remains challenging. Here, the authors present a computational protocol for identifying the specific combination of probe conformations that enables a selective probe–DNA binding mechanism in a recently proposed fluorescent marker, QCy(MeBT)₃, capable of simultaneously recognizing both B-DNA and G-quadruplex DNA through distinct emission signatures.