Twin-FRET: A New Molecular Ruler for Biomolecules

Conformational changes in biomolecules underpin all biological processes and being able to quantify these structural changes in solution is crucial to understand biological function. By carefully positioning two fluorophores within the biomolecule, it is possible to use fluorescence resonance energy transfer (FRET) as a molecular ruler to measure the desired distance. In current FRET assays, the biomolecule needs to be labelled with two different chemical fluorophores acting as a donor-acceptor FRET pair. Incorporation of these two chemically different species at specific positions within the biomolecule is challenging due to limited chemical labelling strategies. Here, we present a radically different strategy for measuring distances in biomolecules. We have developed the concept of TWIN-FRET which removes the need for two different fluorophores attached to the biomolecule by chemically encoding the FRET pair within the structure of the fluorophore itself. We have designed and synthesised a fluorescent molecule to prove this concept. The fluorophore (FH) has an acid-base equilibrium with a ground state pKa∼8.9. We have derivatized the fluorophore to its succinimide ester, and used this derivative, to label a duplex DNA with two molecules of the same fluorophore at specific positions. Our results demonstrate the transfer of non-radiative energy from the neutral (FH) to the anionic (F−) state of the fluorescent molecule. We further demonstrate the use of TWIN-FRET to measure nanometer-size distances within the DNA duplex, and we obtained distance values similar to those obtained using a conventional FRET pair (Alexa488-Cy3). By removing the need to introduce two different chemical structures within the biomolecule, we greatly simplified the methodology to measure nanometer-size distances in biomolecules. We expect this technique to be widely used in structural and biophysical studies of nucleic acids, proteins and interactions between them.