Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Single-Molecule FRET Analyses of NMDA Receptors.

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies.

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide. Combined with Förster resonance energy transfer (FRET) to read out the strain, such peptides represent powerful and versatile nanoscopic force sensing tools. In this paper, we provide a detailed protocol how to synthesize a molecular force sensor for application in T-cell antigen recognition and hands-on guidelines on experiments and analysis of obtained single molecule FRET data.

Fecal proteolytic profiling of pediatric inflammatory bowel disease: A pilot study.

Colonoscopy is the gold standard for diagnosing inflammatory bowel disease (IBD). However, this invasive procedure has a high burden for pediatric patients. Previous research has shown elevated fecal amino acid concentrations in children with IBD versus controls. We hypothesized that this finding could result from increased proteolytic activity. Therefore, the aim of this study was to investigate whether fecal protease-based profiling was able to discriminate between IBD and controls. Protease activity was measured in fecal samples from patients with IBD (Crohn's disease (CD) n = 19; ulcerative colitis (UC) n = 19) and non-IBD controls (n = 19) using a fluorescence resonance energy transfer (FRET)-peptide library. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each FRET-peptide substrate. Screening the FRET-peptide library revealed an increased total proteolytic activity (TPA), as well as degradation of specific FRET-peptides specifically in fecal samples from IBD patients. Based on level of significance (p < .001) and ROC curve analysis (AUC > 0.85), the fluorogenic substrates W-W, A-A, a-a, F-h, and H-y showed diagnostic potential for CD. The substrates W-W, a-a, T-t, G-v, and H-y showed diagnostic potential for UC based on significance (p < .001) and ROC analysis (AUC > 0.90). None of the FRET-peptide substrates used was able to differentiate between protease activity in fecal samples from CD versus UC. This study showed an increased fecal proteolytic activity in children with newly diagnosed, treatment-naïve, IBD. This could lead to the development of novel, noninvasive biomarkers for screening and diagnostic purposes.

Single-Molecule Fluorescence Probes Interactions between Photoactive Protein-Silver Nanowire Conjugate and Monolayer Graphene.

In this work, we apply single-molecule fluorescence microscopy and spectroscopy to probe plasmon-enhanced fluorescence and Förster resonance energy transfer in a nanoscale assemblies. The structure where the interplay between these two processes was present consists of photoactive proteins conjugated with silver nanowires and deposited on a monolayer graphene. By comparing the results of continuous-wave and time-resolved fluorescence microscopy acquired for this structure with those obtained for the reference samples, where proteins were coupled with either a graphene monolayer or silver nanowires, we find clear indications of the interplay between plasmonic enhancement and the energy transfer to graphene. Namely, fluorescence intensities calculated for the structure, where proteins were coupled to graphene only, are less than for the structure playing the central role in this study, containing both silver nanowires and graphene. Conversely, decay times extracted for the latter are shorter compared to a protein-silver nanowire conjugate, pointing towards emergence of the energy transfer. Overall, the results show that monitoring the optical properties of single emitters in a precisely designed hybrid nanostructure provides an elegant way to probe even complex combination of interactions at the nanoscale.

A two-layer circuit cascade-based DNA machine for highly sensitive miRNA imaging in living cells.

Sensitive detection of microRNA (miRNA), one of the most promising biomarkers, plays crucial roles in cancer diagnosis. However, the low expression level of miRNA makes it extremely urgent to develop ultrasensitive and highly selective strategies for quantification of miRNA. Herein, a DNA machine is rationally constructed for amplified detection and imaging of low-abundance miRNA in living cells based on the toehold-mediated strand displacement reaction (TMSDR). The isothermal and enzyme-free DNA machine with low background leakage is fabricated by integrating two DNA circuits into a cascade system, in which the output of one circuit serves as the input of the other one. Once the DNA machine is transfected into breast cancer cells, the overexpressed miRNA-203 initiates the first-layer circuit through TMSDR, leading to the concentration variation of fuel strands, which further influences the assembly of hairpin DNA in the second-layer circuit and the occurrence of fluorescence resonance energy transfer (FRET) for fluorescence imaging. Benefiting from the cascade of the two-layer amplification reaction, the proposed DNA machine acquires a detection limit down to 4 fM for quantification of miR-203 and a 10 000-fold improvement in amplification efficiency over the single circuit. Therefore, the two-layer circuit cascade-based DNA machine provides an effective platform for amplified analysis of low-abundance miRNA with high sensitivity, which holds great promise in biomedical and clinical research.

Photobleaching FRET-FLIM-ICS for quaternary structure quantification on cells. Theory and simulations.

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu.

FLIM-FRET Protein-Protein Interaction Assay.

Fluorescence lifetime imaging performed under FRET conditions between two interacting molecules is a sensitive and robust way to quantify intermolecular interactions in cells. The fluorescence lifetime, an inherent property of the fluorophore, remains unaffected by factors such as concentration, laser intensity, and other photophysical artifacts. In the context of FLIM-FRET, the focus lies on measuring the fluorescence lifetime of the donor molecule, which diminishes upon interaction with a neighboring acceptor molecule. In this study, we present a step-by-step experimental protocol for applying FLIM-FRET to investigate protein-protein interactions involving various RAS isoforms and RAS effectors at the live cell's plasma membrane. By utilizing the FRET pair comprising enhanced green fluorescent protein (eGFP) and fluorescent mCherry, we demonstrate that the proximity and possible nanoclustering of eGFP-tagged KRAS4b G12D and mCherry-tagged KRAS4b WT led to a reduction in the donor eGFP's fluorescence lifetime. The donor lifetime of eGFP-tagged KRAS decreases even further when treated with a dimer-inducing small molecule, or in the presence of RAF proteins, suggesting a greater FRET efficiency, and thus less distance, between donor and acceptor.

KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery.

Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.

Single-Molecule FRET-Based Multiplexed Detection.

Single-molecule multiplexed detection is a high-promise toolkit for the expanding field of biosensing and molecular diagnostics. Among many single-molecule techniques available today for biomarker sensing including fluorescence, force, electrochemical, spectroscopic, barcoding, and other techniques, fluorescence-based approaches are arguably the most widely used methods due to their high sensitivity, selectivity, and readily available fluorophore-labeling schemes for a wide variety of biomolecules. However, multiplexed imaging using fluorescence techniques has proven to be challenging due to the sophisticated labeling schemes often requiring multiple FRET (fluorescence resonance energy transfer) pairs and/or excitation sources, which lead to overlapping signals and complicate data analysis. Here, we describe a single-molecule FRET method that enables multiplexed analysis while still using only one FRET pair, and thus the described approach is a significant step forward from conventional FRET methods.

Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation.

The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.

A molecular dynamics simulation study of glycine/serine octapeptides labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene fluorophore.

The compound 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is a versatile fluorophore widely used in Förster resonance energy transfer (FRET) spectroscopy studies due to its remarkable sensitivity, enabling precise donor-acceptor distance measurements, even for short peptides. Integrating time-resolved and FRET spectroscopies with molecular dynamics simulations provides a robust approach to unravel the structure and dynamics of biopolymers in a solution. This study investigates the structural behavior of three octapeptide variants: Trp-(Gly-Ser)3-Dbo, Trp-(GlyGly)3-Dbo, and Trp-(SerSer)3-Dbo, where Dbo represents the DBO-containing modified aspartic acid, using molecular dynamics simulations. Glycine- and serine-rich amino acid fragments, common in flexible protein regions, play essential roles in functional properties. Results show excellent agreement between end-to-end distances, orientational factors from simulations, and the available experimental and theoretical data, validating the reliability of the GROMOS force field model. The end-to-end distribution, modeled using three Gaussian distributions, reveals a complex shape, confirmed by cluster analysis highlighting a limited number of significant conformations dominating the peptide landscape. All peptides predominantly adopt a disordered state in the solvent, yet exhibit a compact shape, aligning with the model of disordered polypeptide chains in poor solvents. Conformations show marginal dependence on chain composition, with Ser-only chains exhibiting slightly more elongation. This study enhances our understanding of peptide behavior, providing valuable insights into their structural dynamics in solution.

Dual-signal detection of tannic acid in red wines based on the peroxidase activity of carbon dots.

Herein, a novel type of phosphorus and iron-doped carbon dot (P,Fe-CD) with outstanding peroxidase activity and excellent fluorescence performance was hydrothermally synthesized to colorimetrically and fluorimetrically detect tannic acid (TA). In the presence of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, the P,Fe-CDs could oxidize colorless TMB to a blue oxidation product (oxTMB) resulting in an increased value of absorbance. Simultaneously, the fluorescence intensity of P,Fe-CDs at 430 nm could be quenched owing to the fluorescence resonance energy transfer (FRET) between P,Fe-CDs and the generated oxTMB. Meanwhile, after adding the TA to the system containing TMB, H2O2 and P,Fe-CDs, the value of absorbance could be decreased and the fluorescence could be recovered because of the reduction reaction between TA and oxTMB. Therefore, fluorescence intensity and value of absorbance could be applied to quantitatively detect TA with good linearities between the concentration of TA and the fluorescence intensity/value of absorbance (0.997 and 0.997 for the colorimetric signal and fluorimetric one, respectively) and low limits of detection (0.093 µmol L-1 and 0.053 µmol L-1 for the colorimetry and the fluorimetry, respectively), which was successfully applied to the detection of TA in red wines. Moreover, we applied a smartphone-assisted method to the point-of-care detection of TA with accurate results, providing a new technique for TA detection and food quality monitoring.

DNA tetrahedron-based dual-signal fluorescence detection of apoE4 gene sites on a microplate reader.

As a neurodegenerative disorder, Alzheimer's disease (AD) is characterized by cognitive dysfunction and behavioral impairment. Among the various genetic risk factors for AD, apoE4 gene plays a pivotal role in the onset and progression of AD, and detection of apoE4 gene holds significance for prevention and early diagnosis of AD. Herein, dual-signal fluorescence detection of fragments associated with apoE ε4 allele near codon 112 (Tc1) and codon 158 (Tc2) was achieved using DNA tetrahedron nanostructure (DTN). The Förster resonance energy transfer (FRET) process in the DTN was initiated in which the nucleic acid intercalating dye thiazole orange (TO) served as the donor and the cyanine dyes of cyanine3 (Cy3) and cyanine5 (Cy5) at the two vertices of DTN served as the acceptors. In the presence of Tc1 and Tc2, the FRET process between TO and the cyanine dyes was hindered by the enzymatic cleavage reaction, which ensures the dual-signal fluorescence assay of apoE4 gene sites. The limit of detection for Tc1 and Tc2 was estimated to be 0.82 nM and 0.77 nM, respectively, and the whole assay was accomplished within 1 h on a microplate reader. The proposed method thus possesses the advantages of easy operation, short detection time, and high-throughput capability.

Gold nanoclusters-based fluorescence resonance energy transfer for rapid and sensitive detection of Pb2.

Lead pollution has remained a significant global concern for several decades due to its detrimental effects on the brain, heart, kidneys, lungs, and immune system across all age groups. Addressing the demand for detecting trace amounts of lead in food samples, we have developed a novel biosensor based on fluorescence resonance energy transfer (FRET) from fluorescein R6G to gold nanoclusters (AuNCs-CCY). By utilizing polypeptides as a template, we successfully synthesized AuNCs-CCY with an excitation spectrum that overlaps with the emission spectrum of R6G. Exploiting the fact that Pb2+ induces the aggregation of gold nanoclusters, leading to the separation of R6G from AuNCs-CCY and subsequent fluorescence recovery, we achieved the quantitative detection of Pb2+. Within the concentration range of 0.002-0.20 µM, a linear relationship was observed between the fluorescence enhancement value (F-F0) and Pb2+ concentration, characterized by the linear equation y = 2398.69x + 87.87 (R2 = 0.996). The limit of detection (LOD) for Pb2+ was determined to be 0.00079 µM (3σ/K). The recovery rate ranged from 96 % to 104 %, with a relative standard deviation (RSD) below 10 %. These findings demonstrate the potential application value of our biosensor, which offers a promising approach to address the urgent need for sensitive detection of heavy metal ions, specifically Pb2+, in food samples.

A facile dual-mode SERS/fluorescence aptasensor for AFB1 detection based on gold nanoparticles and magnetic nanoparticles.

Aflatoxin B1 (AFB1) is a virulent metabolite secreted by Aspergillus fungi, impacting crop quality and posing health risks to human. Herein, a dual-mode Raman/fluorescence aptasensor was constructed to detect AFB1. The aptasensor was assembled by gold nanoparticles (AuNPs) and magnetic nanoparticles (MNPs), while the surface-enhanced Raman scattering (SERS) and fluorescence resonance energy transfer (FRET) effects were both realized. AuNPs were modified with the Raman signal molecule 4-MBA and the complementary chain of AFB1 aptamer (cDNA). MNPs were modified with the fluorescence signal molecule Cy5 and the AFB1 aptamer (AFB1 apt). Through base pairing, AuNPs aggregated on the surface of MNPs, forming a satellite-like nanocomposite, boosting SERS signal via increased "hot spots" but reducing fluorescence signal due to the proximity of AuNPs to Cy5. Upon exposure to AFB1, AFB1 apt specifically bound to AFB1, causing AuNPs detachment from MNPs, weakening the SERS signal while restoring the fluorescence signal. AFB1 concentration displayed a good linear relationship with SERS/fluorescence signal in the range of 0.01 ng/mL-100 ng/mL, with a detection limit as low as 5.81 pg/mL. The use of aptamer assured the high selectivity toward AFB1. Furthermore, the spiked recovery in peanut samples ranged from 91.4 % to 95.6 %, indicating the applicability of real sample detection. Compared to single-signal sensor, this dual-signal sensor exhibited enhanced accuracy, robust anti-interference capability, and increased flexibility, promising for toxin detection in food safety applications.

AptaShield: A Universal Signal-Transduction System for Fast and High-Throughput Optical Molecular Biosensing.

Biosensing technologies are often described to provide facile, sensitive, and minimally to noninvasive detection of molecular analytes across diverse scientific, environmental, and clinical diagnostic disciplines. However, commercialization has been very limited mostly due to the difficulty of biosensor reconfiguration for different analyte(s) and limited high-throughput capabilities. The immobilization of different biomolecular probes (e.g., antibodies, peptides, and aptamers) requires the sensor surface chemistry to be tailored to provide optimal probe coupling, orientation, and passivation and prevent nonspecific interactions. To overcome these challenges, here we report the development of a solution-phase biosensor consisting of an engineered aptamer, the AptaShield, capable of universally binding to any antigen recognition site (Fab') of fluorescently labeled immunoglobulins (IgG) produced in rabbits. The resulting AptaShield biosensor relies on a low affinity dynamic equilibrium between the fluorescently tagged aptamer and IgG to generate a specific Förster resonance energy transfer (FRET) signal. As the analyte binds to the IgG, the AptaShield DNA aptamer-IgG complex dissociates, leading to an analyte concentration-dependent decrease of the FRET signal. The biosensor demonstrates high selectivity, specificity, and reproducibility for analyte quantification in different biological fluids (e.g., urine and blood serum) in a one-step and low sample volume (0.5-6.25 µL) format. The AptaShield provides a universal signal transduction mechanism as it can be coupled to different rabbit antibodies without the need for aptamer modification, therefore representing a robust high-throughput solution-phase technology suitable for point-of-care applications, overcoming the current limitations of gold standard enzyme-linked immunosorbent assays (ELISA) for molecular profiling.

G-quadruplex modulation by E. coli SSB: A comprehensive study on binding affinities and modes using single-molecule FRET.

G-quadruplexes (GQs) are essential guanine-rich secondary structures found in DNA and RNA, playing crucial roles in genomic maintenance and stability. Recent studies have unveiled GQs in the intergenic regions of the E. coli genome, suggesting their biological significance and potential as anti-microbial targets. Here, we investigated the interaction between homo-tetrameric E. coli SSB and GQ-forming single-stranded DNA (ssDNA) sequence with varying lengths. Combining Microscale Thermophoresis (MST) and conventional spectroscopic techniques, we explored E. coli SSB binding to ssDNA and the structural changes of these secondary DNA structures upon protein binding. Subsequently, we have utilized smFRET to probe the conformational changes of GQ-ssDNA structures upon SSB binding. Our results provide detailed insights into SSB's access to various GQ-ssDNA sequencies and the wrapping of this homo-tetrameric protein around GQ-ssDNA in multiple distinct binding modalities. This study sheds light on the intricate details of E. coli SSB's interaction with ssDNA and the resulting widespread conformational changes within these oligonucleotide structures after protein binding. It offers a thorough insight into SSB's accesses to various GQ-ssDNA architectures. The finding demonstrates the multifaceted binding methods through which this homo-tetrameric protein envelops GQ-ssDNA and could prove valuable in deciphering biological processes that involve DNA G-quadruplexes.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.