Time-resolved Förster Resonance Energy Transfer Research Articles

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer.

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL-1, 0.77 ng∙μL-1, and 1.1 ng∙μL-1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL-1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.

Development and validation of a generic methyltransferase enzymatic assay based on an SAH riboswitch

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) – the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAH. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z’ > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins

G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.

A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW.

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.

A Time‐Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW

AbstractThe peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time‐resolved Förster resonance energy transfer (TR‐FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non‐polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.

Diagnostic TR-FRET assays for detection of antibodies in patient samples

Serological assays are important diagnostic tools for surveying exposure to the pathogen, monitoring immuneresponse post vaccination, and managing spread of the infectious agent among the population.Current serological laboratory assays are often limited because they require the use of specialized laboratory technology and/or work with a limited number of sample types. Here, we evaluate analternative by developing time-resolved Förster resonance energy transfer (TR-FRET) homogeneousassays that exhibited exceptional versatility, scalability, and sensitivity and outperformed or matched currently used strategies in terms of sensitivity, specificity, and precision. We validated the performanceof the assays measuring total immunoglobulin G (IgG) levels; antibodies against severe acute respiratory syndromecoronavirus (SARS-CoV) or Middle Eastern respiratory syndrome (MERS)-CoV spike (S)protein; and SARS-CoV-2S and nucleocapsid (N)proteins and applied it to several large sample sets and real-world applications. We further established a TR-FRET-based ACE2-S competition assay to assess the neutralization propensity of the antibodies. Overall, these TR-FRET-based serological assays can be rapidly extended to other antigens and are compatible with commonly used plate readers.

Defining the structure and dynamics of the N-cTnI-N-cTnC interface in cardiac thin filaments.

The cardiac thin filament (cTF) is a crucial regulator of cardiac muscle contraction. The interface between the N-lobe of TnC and the N-terminus of cTnI (N-cTnI) is known to be a key modulator of calcium binding kinetics. This region contains the serine 23/24 phosphorylation sites, a target of protein kinase A during beta adrenergic signaling. Phosphorylation of this region has shown to produce an increase in calcium dissociation rate in the cTF system. This increase in calcium dissociation rate results in improved diastolic performance to meet hemodynamic demand. The structural mechanism for this observation has yet to be investigated in cTF in-vitro systems. We hypothesize that the N-cTnI contributes to stabilizing the calcium atom in site II via interactions with cTnC and phosphorylation of the serine 23/24 results in the N-cTnI moving away from cTnC allowing for the observed increase in calcium dissociation rate. In order to investigate these proposed structural changes, we performed time-resolved Forster resonance energy transfer (TR-FRET) experiments between cTnI-A28C to cTnC-84C, cTnI-A17C to cTnC-84C, and cTnI-A9C to cTnC-84C in cTF to map N-cTnI with respect to cTnC under different biochemical conditions. We found that N-cTnI is more disordered in the absence of calcium and become more ordered and closer to cTnC upon the addition of calcium. Phosphorylation of the Serine 23/24 sites resulted in an increase in distance and increase in FWHM for the A28C and A17C sites. This increase in distance and disorder observed may explain the increase in calcium dissociation rate as the N-cTnI moves away from cTnC allowing for the rapid release of the calcium atom from cTnC. This hypothesis will be further investigated using molecular dynamics approaches in order to gain atomistic level resolution of specific N-cTnI-N-cTnC interactions.

Diffusion-Enhanced Förster Resonance Energy Transfer in Flexible Peptides: From the Haas-Steinberg Partial Differential Equation to a Closed Analytical Expression.

In the huge field of polymer structure and dynamics, including intrinsically disordered peptides, protein folding, and enzyme activity, many questions remain that cannot be answered by methodology based on artificial intelligence, X-ray, or NMR spectroscopy but maybe by fluorescence spectroscopy. The theory of Förster resonance energy transfer (FRET) describes how an optically excited fluorophore transfers its excitation energy through space to an acceptor moiety-with a rate that depends on the distance between donor and acceptor. When the donor and acceptor moiety are conjugated to different sites of a flexible peptide chain or any other linear polymer, the pair could in principle report on chain structure and dynamics, on the site-to-site distance distribution, and on the diffusion coefficient of mutual site-to-site motion of the peptide chain. However, the dependence of FRET on distance distribution and diffusion is not defined by a closed analytical expression but by a partial differential equation (PDE), by the Haas-Steinberg equation (HSE), which can only be solved by time-consuming numerical methods. As a second complication, time-resolved FRET measurements have thus far been deemed necessary. As a third complication, the evaluation requires a computationally demanding but indispensable global analysis of an extended experimental data set. These requirements have made the method accessible to only a few experts. Here, we show how the Haas-Steinberg equation leads to a closed analytical expression (CAE), the Haas-Steinberg-Jacob equation (HSJE), which relates a diffusion-diagnosing parameter, the effective donor-acceptor distance, to the augmented diffusion coefficient, J, composed of the diffusion coefficient, D, and the photophysical parameters that characterize the used FRET method. The effective donor-acceptor distance is easily retrieved either through time-resolved or steady-state fluorescence measurements. Any global fit can now be performed in seconds and minimizes the sum-of-square difference between the experimental values of the effective distance and the values obtained from the HSJE. In summary, the HSJE can give a decisive advantage in applying the speed and sensitivity of FRET spectroscopy to standing questions of polymer structure and dynamics.

Isothermal Rolling Circle Amplification and Lanthanide‐Based FRET for Femtomolar Quantification of MicroRNA

AbstractQuantification of DNA and RNA biomarkers via isothermal amplification technologies can significantly simplify biosensing and clinical diagnostics. However, highly sensitive and wash‐free assay formats based on time‐resolved Förster resonance energy transfer (TR‐FRET) have been limited to terbium (Tb) based FRET pairs and their application on specialized clinical instruments. Here, we present the implementation of rolling circle amplification (RCA)‐FRET to europium‐dye (Eu‐ATTO620) FRET pairs and its application on standard benchtop fluorescence plate readers. Eu‐ATTO620 RCA‐FRET was used to quantify microRNA‐21 (miR‐21) in a 1 to 100 pM concentration range on a SPARK multimode plate reader system. Detection limits down to 120±20 fM (18±3 attomol of miR‐21) and quantification of endogenous miR‐21 from the plasma of ovarian cancer patients using a KRYPTOR clinical plate reader demonstrated the broad applicability and clinical relevance of Eu‐dye‐based RCA‐FRET. The extension to both Eu and Tb based TR‐FRET and to standard bioanalytical laboratory instruments has the potential to significantly broaden the application and versatility of RCA‐FRET for simple, highly‐sensitive, and multiplexed bioanalysis.

Tiny Titans: Nanobodies as Powerful Tools for TR‐FRET Assay Development

AbstractOwing to their exceptional sensitivity and specificity, assays based on time‐resolved Förster resonance energy transfer (TR‐FRET) have become increasingly popular in biomedical research. TR‐FRET assays are highly versatile and compatible with complex biological environments. However, the paucity of straightforward strategies for the selective installation of TR‐FRET donor fluorophores on target proteins, which is a critical requirement for most assay designs, has limited the potential and adaptation of this powerful technology. To address this issue, we have developed a versatile toolbox strategy based on CoraFluor‐1‐functionalized nanobodies. The target‐agnostic modular design simplifies the TR‐FRET donor‐labeling of epitope tags, fluorescent proteins and primary antibodies, including their use with endogenous proteins in lysates. Our strategy, which we extend to enable the seamless adaptation of split‐luciferase systems for TR‐FRET approaches, greatly facilitates the development and design of TR‐FRET assays for both target engagement studies and the quantification of proteins of interest with sub‐nanomolar sensitivity.

Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility in patient-derived cells.

Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets.

A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader

We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras [PROTACs]) that is capable of both accurately quantifying protein levels in whole-cell lysates in less than 1h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small-molecule inhibitors and PROTACs in a high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation.

Quantification of protein-protein interactions and activation dynamics: A new path to predictive biomarkers

Oncogene dysregulation is a driver of neoplasia development and progression. The use of quantitative molecular imaging to quantify oncogene activation will be crucial in developing companion diagnostics which can identify personalised patient regimens. However, the evaluation of oncogene activation does not necessarily correlate with oncoprotein activation. Post-translational modifications, such as phosphorylation, lipidation and methylation, may enhance oncoprotein functionality. It is this functionality that progresses neoplasia and may correlate with patient outcome. Advanced molecular imaging may be used to directly quantify oncoprotein, as opposed to oncogene, activation. Time-resolved Förster Resonance Energy Transfer (TR-FRET) involves the non-radiative transfer of energy from one chromophore to another over distances of 1-10 nm; allowing FRET to be used as a “chemical ruler”. TR-FRET can be utilised to directly elucidate spatial oncoprotein activation in single cells and patient tissues. In single cells, TR-FRET has uncovered the mechanisms by which PKCβ1 is trafficked to the nucleus and cleaved. Additionally it has revealed the mechanism of activation of Akt/PKB, whereby Akt/PKB undergoes a conformational change, allowing the Thr308 site to be phosphorylated by PDK1. Moreover TR-FRET has been utilised to quantify HER2-HER heterodimerisation and Akt/PKB activation states in patient biopsies, where it is shown to be predictive of outcome/relapse. The role of TR-FRET is not solely limited to intracellular signalling events. A study has used TR-FRET to measure intercellular immune-checkpoint receptor-ligand interactions. Within this study it was seen that PD-L1 expression was not indicative of PD-1/PD-L1 interaction states in a range of solid tumours. Crucially, in melanoma and NSCLC, PD-1/PD-L1 interaction was a predictive of an improved patient outcome. PD-L1 expression did not predict patient outcome. Several groups have worked to improve Fluorescence lifetime imaging microscopy (FLIM) acquisition times, including the use of: window-galvanometers; multifocal multiphoton FLIM and parallel pixel excitation coupled with wide-field time-gated FLIM. The development of novel quantitative molecular imaging will be critical in the development of personalised patient therapies in the future.

Bright and stable luminescent probes for target engagement profiling in live cells.

The pace of progress in biomedical research directly depends on techniques that enable the quantitative interrogation of interactions between proteins and other biopolymers, or with their small-molecule ligands. Time-resolved Förster resonance energy transfer (TR-FRET) assay platforms offer high sensitivity and specificity. However, the paucity of accessible and biocompatible luminescent lanthanide complexes, which are essential reagents for TR-FRET-based approaches, and their poor cellular permeability have limited broader adaptation of TR-FRET beyond homogeneous and extracellular assay applications. Here, we report the development of CoraFluors, a new class of macrotricyclic terbium complexes, which are synthetically readily accessible, stable in biological media and exhibit photophysical and physicochemical properties that are desirable for biological studies. We validate the performance of CoraFluors in cell-free systems, identify cell-permeable analogs and demonstrate their utility in the quantitative domain-selective characterization of Keap1 ligands, as well as in isoform-selective target engagement profiling of HDAC1 inhibitors in live cells.

Abstract MP40: Breakpoint Cluster Region Protein Function As A Kinase Mediates The Atheroprotective Actions Of Hdl In Vascular Endothelium

Introduction: HDL has direct atheroprotective actions on endothelial cells (EC) that include the attenuation of vascular inflammation and promotion of endothelial monolayer integrity. These processes are mediated by scavenger receptor class B, type 1 (SR-BI) and its adaptor protein PDZK1. We have identified the breakpoint cluster region protein (BCR) as a PDZK1 interacting protein in EC. Best known as a gene in the BCR-ABL complex associated with the Philadelphia chromosome, BCR structure predicts both adaptor protein and kinase functions. Hypothesis: Via interaction with PDZK1 and kinase activity, BCR mediates the atheroprotective actions of HDL on EC. Methods: Newly-developed kinase assays that employ time-resolved Forster resonance energy transfer were used to detect kinase activity and identify kinase substrates. In vivo studies were done in ApoAI -/- versus ApoAI -/- ;BCR -/- mice administered control AAV or AAV-ApoAI. Leukocyte-EC interaction and carotid artery reendothelialization were evaluated by intravital microscopy and thermal injury, respectively. Atherosclerosis was invoked using AAV8-PCSK9. BCR expression was compared in human atherosclerotic and normal arteries in two independent publicly available patient cohorts. Results: In cultured EC, HDL activation of eNOS, blunting of EC-monocyte adhesion, and promotion of EC migration all required BCR, and reconstitution studies with BCR mutants revealed requirements for both BCR-PDZK1 interaction and kinase activity. In response to HDL, BCR directly phosphorylated Akt-Ser473 and it provoked Akt-Thr308 phosphorylation by PDK1. ApoAI restoration in vivo in ApoAI -/- mice prompted SR-BI-PDZK1-BCR protein complex formation and BCR phosphorylation of Akt-Ser473 in mouse aorta. It also blunted leukocyte-endothelial cell adhesion and enhanced carotid artery reendothelialization in BCR +/+ , but not in BCR -/- mice. Furthermore, the atheroprotective effect of ApoAI restoration was demonstrable in BCR +/+ , but not in BCR -/- mice. In both human cohorts BCR expression was decreased in atherosclerotic versus normal arteries. Conclusion: BCR function as a novel direct and indirect kinase of Akt mediates the atheroprotective actions of HDL in vascular endothelium.

1,4,9-Triazaspiro[5.5]undecan-2-one Derivatives as Potent and Selective METTL3 Inhibitors.

N6-methyladenosine (m6A) is the most frequent of the 160 RNA modifications reported so far. Accumulating evidence suggests that the METTL3/METTL14 protein complex, part of the m6A regulation machinery, is a key player in a variety of diseases including several types of cancer, type 2 diabetes, and viral infections. Here we report on a protein crystallography-based medicinal chemistry optimization of a METTL3 hit compound that has resulted in a 1400-fold potency improvement (IC50 of 5 nM for the lead compound 22 (UZH2) in a time-resolved Förster resonance energy transfer (TR-FRET) assay). The series has favorable ADME properties as physicochemical characteristics were taken into account during hit optimization. UZH2 shows target engagement in cells and is able to reduce the m6A/A level of polyadenylated RNA in MOLM-13 (acute myeloid leukemia) and PC-3 (prostate cancer) cell lines.

A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept.

ABSTRACTThe ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an “in-house” test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing.

Elucidation of Interactions Between Integration Host Factor and A DNA Four‐Way Junction

Integration host factor (IHF) is an E. coli nucleoid-associated protein that was initially discovered in the integration of bacterial phage DNA. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence and induces a bend of ~160° upon binding. Our previous results have shown that IHF binds to DNA 4WJ that do not contain the consensus sequence with high affinity. DNA Four-Way Junctions (4WJ) are important intermediates in double-strand break repair and homologous recombination. These junctions can adopt either an open or stacked conformation, in which the open conformation facilitates the process of branch migration and strand exchange. In this study, we investigated the IHF-induced changes in the population distribution of junction conformations, using ensemble and single molecule Förster resonance energy transfer (FRET) methods. Our steady state and time-resolved FRET data indicated that under conditions stabilizing the stacked junction conformation, upon binding, IHF induces the open conformation of junction. Single-molecule FRET (smFRET) experiments, performed with total internal reflection fluorescence microscopy, measured population distributions of the junction and showed clear opening of the junctions upon IHF binding. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Cross correlation analyses reveal that the protein slows down the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+, consistent with stabilization of the open conformation. These findings which show that IHF binds to 4WJ with high affinity, induces the open conformation and alters conformational dynamics, suggest that IHF may play multiple roles in the processes of integration and recombination.

TR-FRET Between Engineered Nanobodies Reveals Existence of Endogenous CXCR4 Oligomers

Chemokine receptors CXCR4 and ACKR3 are involved in multiple physiological and pathological processes. In overexpression systems, CXCR4 and ACKR3 form oligomers that are important for chemokine recognition and signaling. Whether this holds true in physiological settings is unknown as no evidence of such oligomers are available. To address this gap of knowledge, we developed a new approach for GPCR oligomer detection by time-resolved Forster resonance energy transfer (TR-FRET) between fluorescently labeled nanobodies. Fluorescent probes were conjugated to the C-terminus of extracellularly binding nanobodies using site-directed sortase-mediated labeling, retaining high affinity of the nanobodies for their target receptors. This approach, first validated in transfected cells by detecting the presence of CXCR4 and ACKR3 homomers and heteromers, reveals for the first time endogenous CXCR4 homomers in non-transfected human leukemia/lymphoma-derived cancer cell lines. More importantly, the investigations in peripheral blood mononuclear cells strongly support the existence of CXCR4 oligomers in native conditions.

A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

Glucagon is a 29-amino acid peptide released from α-cells within pancreatic islets of Langerhans to help raise blood glucose levels. While a plethora of methodologies have been developed for quantitative measurement of insulin released from islets, such methods are not well developed for glucagon despite its importance in blood sugar regulation. In this work, a simple yet robust microfluidic device was developed for holding human pancreatic islets and perfuse them with glucose. The perfusate was collected into 2 min fractions and glucagon quantified using a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay. Simulation of fluid flow within the microfluidic device indicated the device produced low amounts of shear stress on islets, and characterization of the flow with standard glucagon solutions revealed response times within 2 fractions (<4 min). Results with human islets from multiple donors demonstrated either a “burst” of glucagon or a “sustained” glucagon release across the entire period of stimulation. The simplicity, yet robustness, of the device and method is expected to appeal to a number of researchers examining pancreatic islet physiology.