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.