For Förster Resonance Energy Transfer Research Articles

Caged AIEgens: Multicolor and White Emission Triggered by Mechanical Activation.

Aggregation-induced emission luminogens (AIEgens) that respond to mechanical force are increasingly used as force probes, memory devices, and advanced security systems. Most of the known mechanisms to modulate mechanoresponsive AIEgens have been based on changes in aggregation states, involving only physical alterations. Instances that employ covalent bond cleavage are still rare. We have developed a novel mechanochemical uncaging strategy to unveil AIEgens with diverse emission characteristics using engineered norborn-2-en-7-one (NEO) mechanophores. These NEO mechanophores were covalently integrated into polymer molecules and activated in both the solution and solid states. This activation resulted in highly tunable fluorescence upon immobilization through solidification or aggregation, producing blue, green, yellow, and orange-red emissions. By designing the caged and uncaged forms as donor-acceptor pairs for Förster resonance energy transfer (FRET), we achieved multicolor mechanofluorescence, effectively broadening the color spectrum to include white emission. Additionally, we computationally explored the electronic structures of activated NEOs, providing insights into the observed regiochemical effects of the substituents. This understanding, together with the novel luminogenic characteristics of the caged and activated species, provides a highly tunable reporter that traces progress with continuous color evolution. This advancement paves the way for future applications of mechanoresponsive materials in areas like damage detection and bioimaging.

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status.

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

Flexible Förster resonance energy transfer‐assisted optical waveguide based on elastic mixed molecular crystals

AbstractFlexible molecular crystal waveguides based on elastic molecular crystals (EMCs) are essential in flexible and compact optical materials. An increased loss coefficient α due to self‐absorption is often a problem in optical waveguides (OWGs) of fluorescent chemical materials waveguiding photons in active mode. Herein, the development of anthracene‐based elastic mixed molecular crystals (EMMCs) is reported for Förster Resonance Energy Transfer (FRET)‐assisted OWG. To yield a FRET crystal system based on elastic molecular crystals, 1%–5% acceptor doping for fluorescent molecular crystals of 9,10‐dibromoanthracene 1 was successful by selecting the same regioisomer having electron‐withdrawing group, 9,10‐diformylanthracene 2, as a dopant. In addition to conversion to the mixed system, there is a difference in the elastic modulus and hardness in EMC C1 and EMMC C2@1. However, the elastic behaviour was also shown in a few percent doping of the acceptor. The α value of this EMMC, composed of 1 including 1% of 2 (0.0077 dB/μm), is much lower than that of EMC composed of 1 (0.1258 dB/μm) because of reducing self‐absorption in the FRET system. An efficient and flexible OWG was successfully developed by selecting an appropriate acceptor molecule and its low doping rate for mixed crystal construction. This method is a practical approach in various functional and flexible crystal systems.

Relationship between non-photochemical quenching efficiency and the energy transfer rate from phycobilisomes to photosystem II.

The chromophorylated PBLcm domain of the ApcE linker protein in the cyanobacterial phycobilisome (PBS) serves as a bottleneck for Förster resonance energy transfer (FRET)from the PBS to the antennal chlorophyll of photosystem II (PS II) and as a redirection point for energy distribution to the orange protein ketocarotenoid (OCP), which is excitonically coupled to the PBLcm chromophore in the process of non-photochemical quenching (NPQ) under high light conditions. The involvement of PBLcm in the quenching process was first directly demonstrated by measuring steady-state fluorescence spectra of cyanobacterial cells at different stages of NPQ development. The time required to transfer energy from the PBLcm to the OCP is several times shorter than the time it takes to transfer energy from the PBLcm to the PS II, ensuring quenching efficiency. The data obtained provide an explanation for the different rates of PBS quenching in vivo and in vitro according to the half ratio of OCP/PBS in the cyanobacterial cell, which is tens of times lower than that realized for an effective NPQ process in solution.

Compartmentation of cGMP Signaling in Induced Pluripotent Stem Cell Derived Cardiomyocytes during Prolonged Culture.

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β3-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-β-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the β3-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs.

Enhancing Eu3+ red emission in borosilicate glass through UO22+→Eu3+ energy transfer: An actinide acting as sensitizer for lanthanide luminescence

This work projects the feasibility of hexavalent uranium ion as a sensitizer for red emitting europium doped borosilicate glass. Herein we have synthesized the barium borosilicate glass using sol-gel method and characterized the same using x-ray diffraction (XRD) and electron probe micro-analyzer (EPMA). Uranium stabilizes as UO22+ in glass samples endowed by ligand to metal charge transfer (LMCT) displaying unique vibronic coupling fingerprint and green emission. On the other hand europium depicts bright red emission owing to intense 5D0→7F2 electric dipole transitions. Spectral characteristics clearly showed overlap between uranyl emission and Eu3+ absorption paving the way for Förster resonance energy transfer (FRET). Codoping U and Eu in glass triggered UO22+ to Eu3+ energy transfer suggesting red Eu3+ photoluminescence can be effectively sensitized via U6+ ions. Fluorescence decay measurements suggested non-exponential behavior for U6+ singly doped and Eu3+ and U6+ co-doped glass samples with monotonic decrease in average lifetime value of 520 nm emission of uranyl ion with varied doping concentration of Eu3+ ion. At 2.0 mol % Eu3+ion; around 15% UO22+ to Eu3+ energy transfer efficiency was achieved. We believe this work could have long term implications in exploring hexavalent uranium for sensitizer for f-f transitions of lanthanide.

Upconversion FRET quantitation: the role of donor photoexcitation mode and compositional architecture on the decay and intensity based responses

Lanthanide-doped colloidal nanoparticles capable of photon upconversion (UC) offer long luminescence lifetimes, narrowband absorption and emission spectra, and efficient anti-Stokes emission. These features are highly advantageous for Förster Resonance Energy Transfer (FRET) based detection. Upconverting nanoparticles (UCNPs) as donors may solve the existing problems of molecular FRET systems, such as photobleaching and limitations in quantitative analysis, but these new labels also bring new challenges. Here we have studied the impact of the core-shell compositional architecture of upconverting nanoparticle donors and the mode of photoexcitation on the performance of UC-FRET from UCNPs to Rose Bengal (RB) molecular acceptor. We have quantitatively compared luminescence rise and decay kinetics of Er3+ emission using core-only NaYF4: 20% Yb, 2% Er and core-shell NaYF4: 20% Yb @ NaYF4: 20% Yb, 5% Er donor UCNPs under three photoexcitation schemes: (1) direct short-pulse photoexcitation of Er3+ at 520 nm; indirect photoexcitation of Er3+ through Yb3+ sensitizer with (2) 980 nm short (5–7 ns) or (3) 980 nm long (4 ms) laser pulses. The donor luminescence kinetics and steady-state emission spectra differed between the UCNP architectures and excitation schemes. Aiming for highly sensitive kinetic upconversion FRET-based biomolecular assays, the experimental results underline the complexity of the excitation and energy-migration mechanisms affecting the Er3+ donor responses and suggest ways to optimize the photoexcitation scheme and the architecture of the UCNPs used as luminescent donors.

Activators Confined Upconversion Nanoprobe with Near-Unity Förster Resonance Energy Transfer Efficiency for Ultrasensitive Detection

Lanthanide-doped upconversion nanoparticles (UCNPs) as energy donors for Förster resonance energy transfer (FRET) are promising in biosensing, bioimaging, and therapeutic applications. However, traditional FRET-based UC nanoprobes show low efficiency and poor sensitivity because only partial activators in UCNPs possessing suitable distance with energy acceptors (<10 nm) can activate the FRET process. Herein, a novel excited-state energy distribution-modulated upconversion nanostructure is explored for highly efficient FRET. Integration of the optimal 4% Er3+ doped shell and 100% Yb3+ core achieves ∼4.5-fold UC enhancement compared with commonly used NaYF4:20%Yb3+,2%Er3+ nanoparticles, enabling maximum donation of excitation energy to an acceptor. The spatial confinement strategy shortens significantly the energy-transfer distance (∼4.5 nm) and thus demonstrates experimentally a 91.9% FRET efficiency inside the neutral red (NR)-conjugated NaYbF4@NaYF4:20%Yb3+,4%Er3+ nanoprobe, which greatly outperforms the NaYbF4@NaYF4:20%Yb3+,4%Er3+@SiO2@NR nanoprobe (27.7% efficiency). Theoretical FRET efficiency calculation and in situ single-nanoparticle FRET measurement further confirm the excellent energy-transfer behavior. The well-designed nanoprobe shows a much lower detection limit of 0.6 ng/mL and higher sensitivity and is superior to the reported NO2- probes. Our work provides a feasible strategy to exploit highly efficient FRET-based luminescence nanoprobes for ultrasensitive detection of analytes.

New Core-Shell Nanostructures for FRET Studies: Synthesis, Characterization, and Quantitative Analysis.

This work describes the synthesis and characterization of new core-shell material designed for Förster resonance energy transfer (FRET) studies. Synthesis, structural and optical properties of core-shell nanostructures with a large number of two kinds of fluorophores bound to the shell are presented. As fluorophores, strongly fluorescent rhodamine 101 and rhodamine 110 chloride were selected. The dyes exhibit significant spectral overlap between acceptor absorption and donor emission spectra, which enables effective FRET. Core-shell nanoparticles strongly differing in the ratio of donors to acceptor numbers were prepared. This leads to two different interesting cases: typical single-step FRET or multistep energy migration preceding FRET. The single-step FRET model that was designed and presented by some of us recently for core-shell nanoparticles is herein experimentally verified. Very good agreement between the analytical expression for donor fluorescence intensity decay and experimental data was obtained, which confirmed the correctness of the model. Multistep energy migration between donors preceding the final transfer to the acceptor can also be successfully described. In this case, however, experimental data are compared with the results of Monte Carlo simulations, as there is no respective analytical expression. Excellent agreement in this more general case evidences the usefulness of this numerical method in the design and prediction of the properties of the synthesized core-shell nanoparticles labelled with multiple and chemically different fluorophores.

Comparing different modalities of time-resolved two-photon fluorescence measurements for FRET analysis

Different modalities of time-resolved fluorescence lifetime techniques are typically used for Förster resonance energy transfer (FRET) studies in vivo and in solution and have been used to measure macromolecular processes and interactions. Here, we examine the sensitivity of the estimated FRET efficiency and donor-acceptor distance to different modalities of time-resolved two-photon (2P) fluorescence measurements, experimental design, and data analysis. As a model, we used the biosensor (mTurquoise-linker-mCitrine), where mTurquoise-mCitrine serves as the FRET pair tethered with a flexible linker.

Thermoresponsive Self-Assembly of Twofold Fluorescently Labeled Block Copolymers in Aqueous Solution and Microemulsions.

A nonionic double hydrophilic block copolymer with a long permanently hydrophilic and a small thermoresponsive block is synthesized by reversible addition-fragmentation chain-transfer polymerization (RAFT). By employing a specifically designed chain-transfer agent, the polymer is functionalized with complementary end groups which are suited for Förster resonance energy transfer (FRET). The end group attached to the permanently hydrophilic block of poly(N,N-dimethylacrylamide) pDMAm is designed as a permanently hydrophobic segment ("sticker") comprising a long alkyl chain and the 4-aminonaphthalimide fluorophore. The other end attached to the thermoresponsive block of poly(N-isopropylacrylamide) pNiPAm incorporates a coumarin fluorophore. The temperature-dependent self-assembly of the twofold fluorescently labeled copolymer is studied in pure aqueous solution as well as in an o/w microemulsion by several techniques including turbidimetry, dynamic light scattering (DLS), and fluorescence spectroscopy. It is compared to the behaviors of the analogous twofold-labeled pDMAm and pNiPAm homopolymer references. The findings indicate that the block copolymer behaves as a polymeric surfactant at low temperatures, with one relatively small hydrophobic end block and an extended hydrophilic chain forming "hairy micelles". At elevated temperatures above the LCST phase transition of the pNiPAm block, however, the copolymer behaves as an associative telechelic polymer with two nonsymmetrical hydrophobic end blocks, which do not mix. Thus, instead of a network of bridged "flower micelles", large dynamic aggregates are formed. These are connected alternatingly by the original micellar cores as well as by clusters of the collapsed pNiPAm blocks. This type of structure is even more favored in the o/w microemulsion than in pure aqueous solution, as the microemulsion droplets constitute an attractive anchoring point for the hydrophobic dodecyl sticker but not for the collapsed pNiPAm chains.

Realistic Efficiency Limits for Singlet-Fission Silicon Solar Cells.

Singlet fission is a carrier multiplication mechanism that could make silicon solar cells much more efficient. The singlet-fission process splits one high-energy spin-singlet exciton into two lower-energy spin-triplet excitons. We calculated the efficiency potential of three technologically relevant singlet-fission silicon solar cell implementations. We assume realistic but optimistic parameters for the singlet-fission material and investigate the effect of singlet energy and entropic gain. If the transfer of triplet excitons occurs via charge transfer, the maximum efficiency is 34.6% at a surprisingly small singlet energy of 1.85 eV. For the Dexter-type triplet energy transfer, the maximum efficiency is 32.9% at a singlet energy of 2.15 eV. For Förster resonance energy transfer (FRET), the triplet excitons are first transferred into a quantum dot, from which they then undergo FRET into silicon. For this transfer mechanism, the maximum efficiency is 28.% at a singlet energy of 2.33 eV. We show that the efficiency gain from singlet fission is larger the more efficient the silicon base cell is, which stands in contrast to tandem perovskite–silicon solar cells.

Toward the Development of an On-Chip Acoustic Focusing Fluorescence Lifetime Flow Cytometer

Conventional flow cytometry is a valuable quantitative tool. Flow cytometers reveal physical and biochemical information from cells at a high throughput, which is quite valuable for many biomedical, biological, and diagnostic research fields. Flow cytometers range in complexity and typically provide multiparametric data for the user at rates of up to 50,000 cells measured per second. Cytometry systems are configured such that fluorescence or scattered light signals are collected per-cell, and the integrated optical signal at a given wavelength range indicates a particular cellular feature such as phenotype or morphology. When the timing of the optical signal is measured, the cytometry system becomes “time-resolved.” Time-resolved flow cytometry (TRFC) instruments can detect fluorescence decay kinetics, and such measurements are consequential for Förster Resonance Energy Transfer (FRET) studies, multiplexing, and metabolic mapping, to name a few. TRFC systems capture fluorescence lifetimes at rates of thousands of cells per-second, however the approach is challenged at this throughput by terminal cellular velocities. High flow rates limit the total number of photons integrated per-cell, reducing the reliability of the average lifetime as a cytometric parameter. In this contribution, we examine an innovative approach to address this signal-to-noise issue. The technology merges time-resolved hardware with microfluidics and acoustics. We present an “acoustofluidic” time-resolved flow cytometer so that cellular velocities can be adjusted on the fly with a standing acoustic wave (SAW). Our work shows that acoustic control can be combined with time-resolved features to appropriately balance the throughput with the optical signals necessary for lifetime data.

Flexible Nanoporous Liquid Crystal Networks as Matrixes for Förster Resonance Energy Transfer (FRET)

Förster resonance energy transfer (FRET) is important, not only in the fields of biology and biophysics but also in optoelectronics and light guiding systems. Different matrixes are being investigated that facilitate FRET, including zeolites and metal–organic frameworks. In this work, a matrix for FRET generation is proposed: nanoporous liquid crystal networks. These liquid crystal networks can be easily processed and can align dichroic fluorescent dyes. A base treatment can create nanopores in the network, which are then able to absorb a second fluorescent dye in an aqueous phase while still retaining good alignment. Using lifetime measurements, we provide proof that even in this nonoptimized system, around 70% of the energy was transferred via the FRET mechanism from one dye to the other. Liquid crystal networks have many advantages over current matrixes as they are easy to fabricate as well as flexible and could be modified to selectively and reversely absorb dyes, allowing many applications.

Chemoenzymatic Semisynthesis of Phosphorylated α-Synuclein Enables Identification of a Bidirectional Effect on Fibril Formation.

Post-translational modifications (PTMs) impact the pathological aggregation of α-synuclein (αS), a hallmark of Parkinson's disease (PD). Here, we synthesize αS phosphorylated at tyrosine 39 (pY39) through a novel route using in vitro enzymatic phosphorylation of a fragment followed by ligation to form the full-length protein. We can execute this synthesis in combination with unnatural amino acid mutagenesis to include two fluorescent labels for Förster resonance energy transfer (FRET) studies. We determine the effect of pY39 on the aggregation of αS and compare our authentically phosphorylated material to the corresponding glutamate 39 "phosphomimetic." Intriguingly, we find that αS-pY39 can either accelerate or decelerate aggregation, depending on the fraction of phosphorylated protein. The αS-E39 mutant can qualitatively reproduce some, but not all, of these effects. FRET measurements and analysis of existing structures of αS help to provide an explanation for this phenomenon. Our results have important implications for the treatment of PD patients with tyrosine kinase inhibitors and highlight the importance of validating phosphomimetics through studies of authentic PTMs.

Linear and Kinked Oligo(phenyleneethynylene)s as Ideal Molecular Calibrants for Förster Resonance Energy Transfer.

We show that oligo(phenyleneethynylene)s (oligoPEs) are ideal spacers for calibrating dye pairs used for Förster resonance energy transfer (FRET). Ensemble FRET measurements on linear and kinked diads with such spacers show the expected distance and orientation dependence of FRET. Measured FRET efficiencies match excellently with those predicted using a harmonic segmented chain model, which was validated by end-to-end distance distributions obtained from pulsed electron paramagnetic resonance measurements on spin-labeled oligoPEs with comparable label distances.

FRET-Modulated Multihybrid Nanoparticles for Brightness-Equalized Single-Wavelength Barcoding.

Semiconductor quantum dots (QDs) are the most versatile fluorophores for Förster resonance energy transfer (FRET) because they can function as both donors and acceptors for a multitude of fluorophores. However, a complete understanding of multidonor-multiacceptor FRET networks on QDs and their full employment into advanced fluorescence sensing and imaging have not been accomplished. Here, we provide a holistic photophysical analysis of such multidonor-QD-multiacceptor FRET systems using time-resolved and steady-state photoluminescence (PL) spectroscopy and Monte Carlo simulations. Multiple terbium complex (Tb) donors (1-191 units) and Cy5.5 dye acceptors (1-60 units) were attached to a central QD, and the entire range of combinations of FRET pathways was investigated by Tb, QD, and Cy5.5 PL. Experimental and simulation results were in excellent agreement and could disentangle the distinct contributions of hetero-FRET, homo-FRET, and dye dimerization. The FRET efficiency was independent of the number of Tb donors and dependent on the number of Cy5.5 acceptors, which could be used to independently adapt the PL intensity by the number of Tb donors and the PL lifetime by the number of Cy5.5 acceptors. We used this unique tuning capability to prepare Tb-QD-Cy5.5 conjugates with distinct QD PL lifetimes but similar QD PL intensities. These brightness-equalized multihybrid FRET nanoparticles were applied to optical barcoding via three time-gated PL intensity detection windows, which resulted in simple RGB ratios. Direct applicability was demonstrated by an efficient RGB distinction of different nanoparticle-encoded microbeads within the same field of view with both single-wavelength excitation and detection on a standard fluorescence microscope.

A Multicolor Large Stokes Shift Fluorogen‐Activating RNA Aptamer with Cationic Chromophores

Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogues that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO+ , which binds to the Chili aptamer with a low-nanomolar KD . Because of its highly red-shifted fluorescence emission at 592 nm, the Chili-DMHBO+ complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.

Förster Resonance Energy Transfer-Based Dual-Modal Theranostic Nanoprobe for In Situ Visualization of Cancer Photothermal Therapy.

The visualization of the treatment process in situ could facilitate to accurately monitor cancer photothermal therapy (PTT), and dramatically decrease the risk of thermal damage to normal cells and tissues, which represents a major challenge for cancer precision therapy. Herein, we prepare theranostic nanoprobes (NPs) for Förster resonance energy transfer (FRET)-based dual-modal imaging-guided cancer PTT, and clear visualization of the therapeutic process. The FRET-based theranostic NPs exhibit high FRET efficiency (88.2%), good colloidal stability, and tumor-targeting ability. Tumor tissue and surrounding blood vessels are visualized clearly by FRET-based NIR fluorescence imaging with a high signal-to-background ratio (14.5) and photoacoustic imaging with an excellent resolution at 24 h post injection of NPs. Under the guidance of dual-modal imaging, the NPs-induced photothermal effect selectively destructs cancer cells, simultaneously decreasing the FRET efficiency and leading to fluorescence and photoacoustic signal changes. The sensitive self-feedback process enables the in situ visualization of therapeutic process and precision guidance of in vivo cancer PTT. A high therapeutic efficacy and minimum side effects are achieved in C6 tumor-bearing nude mice, holding great promise for precision therapy and cancer theranostics.

Using a Specific RNA-Protein Interaction To Quench the Fluorescent RNA Spinach.

RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. "Spinach" is a genetically encodable RNA aptamer that starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyze RNA-protein interactions by quenching the spinach aptamer.