Forster resonance energy transfer for studying nucleic acids denaturation: A chemical and biological sciences laboratory experiment

Abstract

The Förster resonance energy transfer (FRET) melting assay intends to evaluate the unfolding, denaturation process of DNA secondary structures, and its stabilization using compounds known as DNA binders, some of which are highly specific for G‐quadruplex DNAs versus duplex DNAs. First, students determined the melting temperature (Tm) of DNA sequences double labeled with 5′‐FAM (fluorescein) and 3′‐TAMRA (tetramethylrhodamine) in the absence of DNA binders. Second, they determined the melting temperature of the DNAs in the presence of DNA binders by monitoring fluorescence. After completing this experiment, students understood that this method allows a semiquantitative analysis to test a variety of DNA binders against DNA secondary structures, and it can be used to rapidly identify the most promising drug candidates in the drug development stages at the basic research level.

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

“…The technique is highly suitable to evaluate the changes in the G4 folding and unfolding processes ( Figure 20 ). In FRET experiments, G4 oligonucleotides are labeled at the 5′- and 3′-end with a donor and acceptor fluorophore, respectively [ 194 ]. Briefly, the excited state energy is transferred from the donor to the acceptor via dipole–dipole interactions.…”

Structural Insights into tRNA via Hypochromism

“…Additionally, this structure disruption can be monitored via the hyperchromic effect, an increase in A 260 which occurs upon loss of the duplex element. Experimental protocols have been published describing how either the intrinsic change in A 260 or change in fluorescence from labeled strands can be observed as a function of temperature to monitor DNA duplex melting and obtain thermodynamic values for the process. − A drawback of this approach is it requires sophisticated instrumentation with temperature control features and limits the number of students who can complete an entire melting curve within a typical lab period. As a “work-around” to the drawbacks of the aforementioned experiments, an experimental protocol was recently published that allows DNA duplex melting curves to be obtained without sophisticated instrumentation .…”