Ongoing research into intracellular reduction/oxidation (redox) systems is creating opportunities for understanding and manipulating the molecular basis of cancer. The redox environment contributes in significant ways to a range of cellular processes — for example, DNA synthesis, enzyme activation, selective gene expression, and cell proliferation, differentiation and apoptosis. Having the ability to sense the environment can provide insight into, and can help to control, those processes.Investigators are working to develop genetically encoded biosensors with which to monitor alterations in intracellular redox potentials in real time without disrupting the cells. Förster resonance energy transfer (FRET) offers one possibility. In a FRET-based biosensor, a redox event would lead to a conformational change in the sensor, which in turn would induce a measurable change in the distance between the donor and acceptor molecules. However, this option suffers from a lack of redox-sensitive linkers separating the donor from the acceptor molecules. In the February issue of Experimental Biology and Medicine, researchers at the University of Illinois at Urbana-Champaign reported the development of chimeric peptides that can serve as linkers for FRET-based biosensors. Researchers have developed chimeric peptides that can act as linkers for FRET-based biosensors. Each linker is an α-helix in its reduced state. Disulfide bonds form upon oxidation and shift the free energy minimum from the α-helix to a “clamped coil” state, bringing the two fluorescent proteins (ECFP and EYFP) closer to one another and thus enabling FRET. © Society for Experimental Biology and Medicine.“We designed a series of redox-sensitive linkers that undergo conformational changes upon oxidation/reduction and, through a series of synthesis/FRET experiments, selected the most promising designs,” said Robert M. Clegg, one of the study’s authors. The system involves peptide linker sequences, Clegg said, in which, in their reduced states, the linkers are α-helices. Thiol groups positioned throughout each linker sense the redox potential of the environment and, upon oxidation, form disulfide bonds. Under oxidative conditions, these bonds shift the free energy minimum from the α-helix to a “clamped coil” state, bringing the two fluorescent proteins closer to one another and thus enabling FRET. The investigators tested the linkers by attaching them to a widely used FRET-compatible pair of GFP variants, enhanced cyan fluorescent protein (CFP) and enhanced yellow fluorescent protein (YFP), and validated them in vitro and in mammalian cell cultures. The researchers performed the in vitro fluorescence measurements with a modified photon-counting fluorometer and acquired fluorescence emission spectra from 460 to 750 nm while exciting the CFP at 440 nm, and from 520 to 750 nm while exciting the YFP at 500 nm. They determined FRET efficiencies using a ratiometric method referred to as (ratio)A. In this technique, (ratio)A is the ratio between the acceptor fluorescence (which occurs because of energy transfer from the excited donor) and the directly excited acceptor fluorescence. Increased energy transfer results in greater emission intensity from the acceptor molecules and consequently in a higher (ratio)A value. In addition to validating the linkers for FRET-based biosensors, the study demonstrated the efficacy of using the (ratio)Amethod with linked GFP variants. The technique has been shown to be reproducible, the researchers explained, because it automatically corrects for sample-to-sample variation in fluorophore concentration and because it automatically cancels changes in the acceptor fluorophores’ fluorescence quantum yield resulting from alterations in the acceptor chromophores’ microenvironment. Development of FRET-based biosensors for measuring oxidative stress in living cells and tissues could benefit biomedical researchers in a variety of areas. Such advancement is especially important, Clegg said, given the significant role of the intracellular redox state in determining a cell’s fate and given the mounting evidence that perturbations in the redox state are associated with cancer, with various inflammatory disorders and with aging. FRET-based redox sensors thus hold promise for understanding a range of molecular mechanisms underlying human health and disease. The FRET-based method offers several advantages over other redox-sensitive biosensors currently under development. A recently reported redox-sensitive GFP exhibits various steady-state fluorescence intensities, depending on the redox conditions, for example. However, with this technique, researchers must consider the ratio of two images acquired at two different excitation wavelengths. Also, they must align multiple laser lines as well as continually monitor and calibrate their relative intensities. “Methods based on monitoring steady-state fluorescence intensities of a single fluorescence component are sometimes difficult to quantify compared to a FRET-based sensor for live-cell and tissue imaging,” Clegg said. “For FRET, the increase in the acceptor fluorescence can only take place if there is a change in the efficiency of energy transfer. This specificity and discriminatory ability of FRET is one of the driving motives behind our development of a FRET-based assay, instead of relying only on changes in the fluorescence intensity of a single component.” The researchers are continuing to develop the FRET-based biosensors. Currently, upon oxidation, their best sensor shifts the FRET efficiency from approximately five to 30 percent — a very large response, Clegg said. Also, they have added fluorescence lifetime imaging to the FRET measurements, which, according to Clegg, offers several advantages over simple fluorescence intensity measurements: For example, it is generally better at rejecting scattered light and background fluorescence. This will be especially helpful when applying FRET-based redox biosensors to biomedical imaging research, he said.Contact: Robert M. Clegg, University of Illinois at Urbana-Champaign; e-mail: rclegg@uiuc.edu.