Evaluating the Relationship between FRET Changes and Distance Changes Using DNA Length and Restriction Enzyme Specificity

Abstract

FRET (Forster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students measured the apparent FRET between donor and acceptor fluorophores located on the ends of several DNAs of unknown lengths, enabling them to order the DNAs according to size. In addition, students investigated site-specific DNA cleavage by restriction endonucleases, using loss of apparent FRET to determine which enzyme cut sites were present in each of the DNAs. After completing this experiment, students understood the inverse relationship between changes in FRET and changes in distance, and understood how changes in FRET could be used to monitor a conformational change in a molecule. As an extension to the experiment, a tutorial is included that uses the same DNAs to illustrate the ability of singlemolecule FRET measurements to resolve heterogeneity in a sample, which cannot be done via more traditional ensemble measurements.

Open-Source Fluorescence Spectrometer for Noncontact Scientific Research and Education

“…Fluorescence spectral analysis is extensively used to measure multiple fluorescent compounds in sample solutions in diverse fields ranging from biomedical applications to environmental monitoring. − Förster resonance energy transfer or fluorescence resonance energy transfer (FRET) requires spectral measurements to quantify the relative intensity between donor and acceptor fluorophores and enables the real-time analysis of molecular structure changes and interactions by accurately measuring molecular proximity. − Multiple fluorescence colors of quantum dots need to be quantified for multiplexed assays that exploit the unique optical characteristics of fluorescent nanocrystals, such as simultaneous excitation for multiple fluorescence colors and size-tunable emission wavelengths. − Fluorescence spectroscopic characterization of algal and cyanobacterial cells provides a powerful method to discriminate problematic species and predict algal blooms. − Fluorescence spectroscopy is a highly versatile and widely used technique for these applications; however, commercial spectrometers remain bulky, delicate, and expensive, which limits their use to well-equipped laboratories.…”

Designing a Compact, Low-Cost FRET Microscopy Platform for the Undergraduate Classroom

“…Förster resonance energy transfer (FRET)based sensors (10)(11)(12)(13) are still the most prevalent and diverse, having been leveraged to measure protein-protein interactions (14)(15)(16), small molecules (17)(18)(19)(20), enzyme activity (21)(22)(23)(24), signal transduction, motility, and other cellular processes (25)(26)(27)(28). Despite the accessibility of nonprofit plasmid repositories for FRET probes (e.g., Addgene) and the widespread agreement that STEM training should begin early (29)(30)(31)(32)(33), FRET imaging is not yet widely integrated in undergraduate laboratory curricula, with only a handful of descriptions in educational journals (34)(35)(36)(37).…”

Chemical cross‐linking of a variety of green fluorescent proteins as Förster resonance energy transfer donors for Yukon orange fluorescent protein: A project‐based undergraduate laboratory experience

“…While phosphofructokinase may have served as the best protein for fluorescence studies involving FRET in the 1990s, the ease of obtaining GFPs with a wide range of visible emission now make GFPs the ideal protein for fluorescent studies of FRET. Despite the wide range of applications of GFPs in FRET-based research applications [13][14][15][16][17][18][19][20], to the best of our knowledge, only three experiments using FRET are described in biochemistry-related educational journals [10,21,22], and there are no published undergraduate experiments which explore FRET using GFPs.…”

“…FRET, the process by which the energy of an excited donor molecule is nonradiatively transferred to an acceptor molecule, is a common topic taught in biochemistry, physical chemistry, and biophysics. Approaches to teaching this topic in the laboratory setting include conformational stability of proteins and evaluating the interaction between DNA and restriction enzymes . Royer discussed the many ways in which one can use fluorescence spectroscopy as a teaching tool for FRET, emphasizing the study of the fluorescence properties of the single tryptophan residue in phosphofructokinase as the model example .…”