Förster Resonance Energy Transfer Acceptor Research Articles

Quantitative FRET (qFRET) Technology for the Determination of Protein-Protein Interaction Affinity in Solution.

Protein–protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research to detect molecular interactions in vitro and in vivo. The FRET assay provides very high sensitivity and efficiency. Several attempts have been made to develop the FRET assay into a quantitative measurement for protein–protein interaction affinity in the past. However, the progress has been slow due to complicated procedures or because of challenges in differentiating the FRET signal from other direct emission signals from donor and receptor. This review focuses on recent developments of the quantitative FRET analysis and its application in the determination of protein–protein interaction affinity (KD), either through FRET acceptor emission or donor quenching methods. This paper mainly reviews novel theatrical developments and experimental procedures rather than specific experimental results. The FRET-based approach for protein interaction affinity determination provides several advantages, including high sensitivity, high accuracy, low cost, and high-throughput assay. The FRET-based methodology holds excellent potential for those difficult-to-be expressed proteins and for protein interactions in living cells.

Photomodulation of a Dual‐Color Luminescent System Combining Quantum Dots with a FRET Acceptor Ligand**

AbstractHerein, we report on a dual‐color, photoswitchable donor‐acceptor nanosystem based on quantum dots (QD) and an anthryl quinolyl ethylene photoactive functional ligand (AQE‐FL), in which a carboxylic group is used to anchor AQE‐FL to QD. Photophysical and photochemical properties of the resulting photoswitchable QD system (psQD) were studied by steady state and time‐resolved spectroscopic methods. Efficient Förster resonance energy transfer (FRET) from QD to AQE‐FL was shown. Applicability of the Stern‐Volmer formalism (with a subdivision into static and dynamic quenching) to the psQD studied is discussed. The spectral overlap integrals of QD emission and trans‐ and cis‐AQE‐FL isomers absorption differ by 3 times that which is used for controlling the FRET efficiency. Consequently, the psQD emission is redistributed between the QD (417 nm) and AQE (520 nm) channels, i. e. dual‐color fluorescent switching between two observable states without the “off” state is recorded.

Improving the flexibility of genetically encoded voltage indicators via intermolecular FRET

A new family of genetically encoded voltage indicators (GEVIs) has been developed based on intermolecular Förster resonance energy transfer (FRET). To test the hypothesis that the GEVI ArcLight functions via interactions between the fluorescent protein (FP) domains of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were cotransfected with both the FRET donor and acceptor GEVIs. Using a cyan fluorescent protein donor and an RFP acceptor, we were able to observe a voltage-dependent signal with an emission peak separated by over 200 nm from the excitation wavelength. The intermolecular FRET strategy also works for rhodopsin-based probes, potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. The signals are larger because the voltage-induced conformational change moves two FPs independently. The expression of the FRET donor and acceptor can also be restricted independently, enabling greater cell type specificity as well as refined subcellular voltage reporting.

Erythrocyte membrane-camouflaged carrier-free nanoassembly of FRET photosensitizer pairs with high therapeutic efficiency and high security for programmed cancer synergistic phototherapy

Phototherapy has been intensively investigated as a non-invasive cancer treatment option. However, its clinical translation is still impeded by unsatisfactory therapeutic efficacy and severe phototoxicity. To achieve high therapeutic efficiency and high security, a nanoassembly of Forster Resonance Energy Transfer (FRET) photosensitizer pairs is developed on basis of dual-mode photosensitizer co-loading and photocaging strategy. For proof-of-concept, an erythrocyte-camouflaged FRET pair co-assembly of chlorine e6 (Ce6, FRET donor) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR, FRET acceptor) is investigated for breast cancer treatment. Notably, Ce6 in the nanoassemby is quenched by DiR and could be unlocked for photodynamic therapy (PDT) only when DiR is photobleached by 808-nm laser. As a result, Ce6-caused phototoxicity could be well controlled. Under cascaded laser irradiation (808–660 nm), tumor-localizing temperature rise following laser irradiation on DiR not only induces tumor cell apoptosis but also facilitates the tumor penetration of NPs, relieves tumor hypoxia, and promotes the PDT efficacy of Ce6. Such FRET pair-based nanoassembly provides a new strategy for developing multimodal phototherapy nanomedicines with high efficiency and good security.

Smartphone-assisted detection of nucleic acids by light-harvesting FRET-based nanoprobe

Point-of-care assays for optical detection of biomolecular markers attract growing attention, because of their capacity to provide rapid and inexpensive diagnostics of cancer and infectious diseases. Here, we designed a nanoprobe compatible with a smartphone RGB camera for detection of nucleic acids. It is based on light-harvesting polymeric nanoparticles (NPs) encapsulating green fluorescent donor dyes that undergo efficient Förster Resonance Energy Transfer (FRET) to red fluorescent acceptor hybridized at the particle surface. Green-emitting NPs are based on rhodamine 110 and 6G dyes paired with bulky hydrophobic counterions, which prevent dye self-quenching and ensure efficient energy transfer. Their surface is functionalized with a capture DNA sequence for cancer marker survivin, hybridized with a short oligonucleotide bearing FRET acceptor ATTO647N. Obtained 40-nm poly(methyl methacrylate)-based NP probe, encapsulating octadecyl rhodamine 6G dyes with tetrakis(perfluoro-tert-butoxy)aluminate counterions (~6000 dyes per NP), and bearing 65 acceptors, shows efficient FRET with >20% quantum yield and a signal amplification (antenna effect) of 25. It exhibits ratiometric response to the target DNA by FRET acceptor displacement and enables DNA detection in solution by fluorescence spectroscopy (limit of detection 3 pM) and on surfaces at the single-particle level using two-color fluorescence microscopy. Using a smartphone RGB camera, the nanoprobe response can be readily detected at 10 pM target in true color and in red-to-green ratio images. Thus, our FRET-based nanoparticle biosensor enables detection of nucleic acid targets using a smartphone coupled to an appropriate optical setup, opening the way to simple and inexpensive point-of-care assays.

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor.

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.

A Tumor‐Microenvironment‐Responsive Lanthanide–Cyanine FRET Sensor for NIR‐II Luminescence‐Lifetime In Situ Imaging of Hepatocellular Carcinoma

Deep tissue imaging in the second near-infrared (NIR-II) window holds great promise for widespread fundamental research. However, inhomogeneous signal attenuation due to tissue absorption and scattering hampers its application for accurate in vivo biosensing. Here, lifetime-based in situ hepatocellular carcinoma (HCC) detection in NIR-II region is presented using a tumor-microenvironment (peroxynitrite, ONOO- )-responsive lanthanide-cyanine Förster resonance energy transfer (FRET) nanosensor. A specially designed ONOO- -responsive NIR-II dye, MY-1057, is synthesized as the FRET acceptor. Robust lifetime sensing is demonstrated to be independent of tissue penetration depth. Tumor lesions are accurately distinguished from normal tissue due to the recovery lifetime. Magnetic resonance imaging and liver dissection results illustrate the reliability of lifetime-based detection in single and multiple HCC models. Moreover, the ONOO- amount can be calculated according to the standard curve.

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

Comparison of spectral FRET microscopy approaches for single-cell analysis.

Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Z-resolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET "controls" with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.

Diverse Applications of Intermolecular FRET in Voltage Imaging

Genetically encoded voltage indicators (GEVIs) enables the optical measurement of changes in membrane potential by a change in the fluorescence of the fluorescent protein (FP). ArcLight, one of the most popular GEVIs, is structurally composed of three primary domains: four transmembrane segments that constitutes the voltage-sensing domain (VSD), a cytoplasmic linker region that fuses a FP to the VSD, and the cytoplasmic FP, which functions as the optical reporter. Modifications to any of the domains results in an alteration of the GEVI optical signal. Intermolecular Förster Resonance Energy Transfer (FRET) was used to study the interactions between GEVI proteins when modifications were made to the above-mentioned domains. Robust voltage-dependent signals were observed from GEVIs utilizing either CFP-YFP or GFP-RFP FRET pairs. Mutations to the FPs results in changes of the voltage-dependent FRET signal, suggesting a change in interaction, which may result in a change of the distance and/or orientation of the donor and acceptor FPs. Attempts were also made to identify the optimal linker length for the best FRET interaction between the GEVIs. Intermolecular FRET also enables for the study of specific areas of the neuron. For instance, an axon targeted FRET donor can be co-expressed with a GEVI fused with the FRET acceptor. Only specific areas that expresses both the FRET donor and acceptor FP will result in a FRET signal. The many applications of intermolecular FRET create a wide range of possibilities ranging from better cell-type specific expression to better 2-photon probes.

MiRNA-21 rapid diagnosis by one-pot synthesis of highly luminescent red emissive silver nanoclusters/DNA

microRNAs (miRNAs) are significant biomarkers either for probing cellular events or disease diagnosis. Compared to regular RNA, miRNAs possess short length and low abundance with sequence homology, which results in major challenges on the determination of these biomarkers. Thus, developing a simple, rapid, and effective technique for qualitative and quantitative analysis of miRNAs is urgent in clinical diagnosis, pathogenesis, and various medical therapies.Herein, DNA-silver nanoclusters (DNA/AgNCs) have been synthesized by a specific DNA scaffold containing a cytosine enriched fragment and a capitation agent with high selectivity probe, to design a Förster resonance energy transfer (FRET) sensing platform for miRNA-21 detection. A duplex miRNA-21/DNA probes structure is formed by introducing a red-emitting synthesized DNA/AgNCs, as a FRET donor, to miRNA-21 in the presence of a near-infrared (NIR)-emitting probe-modified Cy5.5, as a FRET acceptor. These sequences produced a duplex structure which could be utilized as a bridge for the successful transferring of the energy from DNA/AgNCs excited to Cy5.5 at steady state. This hybridized structure could result in the enhancement of Cy5.5 fluorescence intensity in a linearly proportional manner toward miRNA-21 concentration as a target. We believe this as-designed FRET-based technique owning high selectivity, low detection limit (4.0 × 10−3 nM), and a wide dynamic ranges of 0.02–100.0 nM, can be introduced as a new advanced method to develop specific miRNAs-based clinical diagnoses.

Synthesis of Functionalized Silica Particles for Label‐free Detection of PTP1B Using FRET

Silica nanoparticles (SiNPs, 30 and 70 nm) and microparticles (SiMPs, 10 μm) were functionalized with a PTP1B specific probe for label‐free fluorescence detection of PTP1B. Intrinsic tryptophan residues of PTP1B serve as the Förster resonance energy transfer (FRET) donor and the functionalized silica particles as the FRET acceptor. When a mixture of the functionalized SiNP and PTP1B is excited at tryptophan excitation maximum (280 nm), significant sensitized fluorescence was detected at 450 nm as a result of FRET. The limit of detection of PTP1B was found to be 29 and 70 nM for the functionalized 30 and 70 nm SiNP, respectively. Using this approach, PTP1B could be detected in E. coli cell lysate in which PTP1B was induced. The functionalized SiNP probe (SiNP/NA) was selective toward PTP1B over BSA, and the nanoparticles are able to penetrate into cells. Furthermore, the binding of PTP1B to the SiMPs could be visualized under a deep UV‐microscope.

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe.

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.

Probing direct interaction of oncomiR-21-3p with the tumor suppressor p53 by fluorescence, FRET and atomic force spectroscopy

miRNA-21-3p is overexpressed in a number of cancers and contributes to their development with a concomitant inhibition of the p53 onco-suppressive function. While a direct interaction of p53 with some miRNA precursors (namely pri-miRNAs and pre-miRNAs) was found, no interaction with mature micro RNA has been so far evidenced. It could therefore be very interesting to investigate if a direct interaction of miR-21-3p and p53 is occurring with possible impairment of the p53 onco-suppressive function. Fluorescence and Atomic Force Spectroscopy (AFS) were applied to study the interaction of p53 DNA Binding Domain (DBD) and miRNA-21-3p. Förster resonance energy transfer (FRET) was used to measure the distance between the DBD lone tryptophan (FRET donor) and a dye (FRET acceptor) bound to miRNA-21-3p. AFS and Fluorescence evidenced a direct interaction between miRNA-21-3p and DBD; with the formed complex being characterized by an affinity of 105 M, with a lifetime in the order of seconds. FRET allowed to determine an average distance of 4.0 nm between the DBD lone Trp146 and miRNA-21-3p; consistently with the involvement of the DBD L3 loop and/or the H1 helix in the complex formation, directly involved in the oligomerization and DNA binding. This may suggest that a functional inhibition of p53 could arise from its interaction with the oncogenic miRNA. Evidence of DBD-miRNA-21-3p complex formation may deserve some interest for inspiring novel therapeutic strategies.

Construction of the FRET Pairs for the Visualization of Mitochondria Membrane Potential in Dual Emission Colors.

Mitochondria membrane potential (MMP) play significant roles during metabolism, signaling, and other important bioevents. Visualization of MMP levels is essential for many biological researches. However, fluorescent probes for monitoring MMP levels in dual emission colors are still deficient, which greatly limited the development of relative research areas. In this work, a pair of fluorescent probes have been designed and synthesized to monitor the MMP levels in dual emission colors based on Forster resonance energy transfer (FRET) mechanism. The FRET donor (FixD) is constructed by linking a benzyl chloride group to a fluorophore with bright-green emission. The FixD could target mitochondria and be immobilized in mitochondria by linking to the thiol group of mitochondrial proteins. The FRET acceptor (LA) is designed with green absorption and deep-red emission. In live cells with high MMP levels, FixD and LA both target mitochondria, and deep-red (DR) emission could be detected with the excitation of 405 nm. Particularly, the spectral shift of fluorescence upon the decrease of MMP is up to 110 nm, which is greatly favorable for the clear observation of MMP levels. With the decrease of MMP, LA would be released from mitochondria while FixD would still be immobilized in mitochondria, and decreased DR emission and increased green fluorescence could be detected due to the absence of FRET. In this manner, the MMP levels could be monitored in dual emission colors.

Development of Novel Dark Quenchers and Their Application to Imaging Probes

Rhodamine dyes are among the most widely used fluorescent dyes for bioimaging due to their high fluorescence quantum yield and high photostability. Recently, novel far-red to near-infrared (NIR) fluorescent dyes have been developed: Si-rhodamines (SiRs), in which the O atom of conventional rhodamine dyes at the 10 position of the xanthene moiety is replaced with a Si atom. These SiRs are excellent long-wavelength fluorophores for bioimaging, as they retain the advantageous photophysical properties of conventional rhodamine dyes. Further, we focused on the QSY dark quenchers, which contain the rhodamine scaffold bearing aromatic rings at the N atoms at the 3,6-positions of the xanthene moiety; these show no fluorescence, irrespective of solvent polarity and pH. NIR fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). We successfully developed SiR-based NIR dark quenchers (SiNQs) which show broad absorption covering this region. To demonstrate their usefulness, we designed and synthesized a NIR fluorescence probe for matrix metalloproteinase (MMP) activity using SiNQs. The developed probe was able to detect MMP activity in terms of NIR fluorescence, not only in vitro, but also in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized.

The Development of Intrinsically Fluorescent Unnatural Amino Acids for In Vivo Incorporation into Proteins

The amino acid acridonylalanine (Acd) can be a valuable tool for studying protein dynamics. On its own, Acd's long fluorescence lifetime of 15 ns has been beneficial for polarization experiments to reveal protein binding dynamics in biological pathways. Acd can also reveal structural dynamics as part of a Förster resonance energy transfer (FRET) or photoinduced electron transfer (eT) probe pair. We have reported successful, selective genetic incorporation of Acd by an aminoacyl tRNA synthetase. This allows us to site-specifically label a protein of interest expressed in E. coli with minimal perturbation. While Acd is valuable in itself, we are developing on a series of Acd derivatives that can shift Acd's fluorogenic properties while still being ribosomally permitted for genetic incorporation. We have shown that we can red shift Acd's absorption and emission spectra and modulate it's brightness. One derivative has been shown be an enhanced FRET acceptor in a peptide cleavage assay and can be activated by the aminoacyl tRNA synthetase for genetic incorporation. Our long-term goal is to develop FRET pairs that can be completely genetically encoded to enable monitoring of protein processes that cannot be accessed with other labeling methods, such as real-time monitoring of co-translational protein folding.

Tuning the interfacial and energetic interactions between a photoexcited conjugated polymer and open-shell small molecules.

Design rules and application spaces for closed-shell conjugated polymers have been well established in the field of organic electronics, but the emerging class of open-shell stable radicals has not been evaluated in such detail. Thus, establishing the underlying physical phenomena associated with the interactions between both classes of molecules is imperative for the effective utilization of these soft materials. Here, we establish that Förster Resonance Energy Transfer (FRET) is the dominant mechanism by which energy transfer occurs from a common conjugated polymer to various radical species using a combination of experimental and computational approaches. Specifically, we determined this fact by monitoring the fluorescence quenching of poly(3-hexylthiophene) (P3HT) in the presence of three radical species: (1) the galvinoxyl; (2) the 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO); and (3) the 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radicals. Both in solution and in the solid-state, the galvinoxyl and PTIO radicals showed quenching that was on par with that of a common fullerene electron-accepting derivative, due to the considerable overlap of their absorbance spectrum with the fluorescence spectrum of the P3HT species, which indicated that isoenergetic electronic transitions existed for both species. Conversely, TEMPO showed minimal quenching at similar concentrations due to the lack of such an overlap. Furthermore, computational studies demonstrated that FRET would occur at a significantly faster rate than other competing processes. These findings suggest that long-range energy transfer can be accomplished in applications when radicals that can act as FRET acceptors are utilized, forming a new design paradigm for future applications involving both closed- and open-shell soft materials.

Interaction of the anticancer p28 peptide with p53-DBD as studied by fluorescence, FRET, docking and MD simulations

BackgroundThe p28 peptide, derived from the blue copper protein Azurin, exerts an anticancer action due to interaction with the tumor suppressor p53, likely interfering with its down-regulators. Knowledge of both the kinetics and topological details of the interaction, could greatly help to understand the peptide anticancer mechanism. MethodsFluorescence and Förster resonance energy transfer (FRET) were used to determine both the binding affinity and the distance between the lone tryptophan (FRET donor) of DNA Binding Domain (DBD) of p53 and the Iaedens dye (FRET acceptor) bound to the p28 peptide. Docking, Molecular Dynamic simulations and free energy binding calculations were used to single out the best complex model, compatible with the distance measured by FRET. ResultsTryptophan fluorescence quenching provided a 105 M−1 binding affinity for the complex. Both FRET donor fluorescence quenching and acceptor enhancement are consistent with a donor-acceptor distance of about 2.6 nm. Docking and molecular dynamics simulations allowed us to select the best complex, enlightening the contact regions between p28 and DBD. Conclusionsp28 binds to DBD partially engaging the L1 loop, at the same region of the p53 down-regulator COP1, leaving however the DNA binding site available for functional interactions. General significanceElucidation of the DBD-p28 complex gets insights into the functional role of p28 in regulating the p53 anticancer activity, also offering new perspectives to design new drugs able to protect the p53 anticancer function.

Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains.

A flexible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large Förster radii of traditional FRET methods (R0 > 20 Å) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-l-fluorotryptophan, or tyrosine. The Förster radii are always close to 10 Å, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative fluorescence lifetime of the donor and the diffusion coefficient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.