Förster resonance energy transfer biosensors for fluorescence and time-gated luminescence analysis of rac1 activity.

Genetically encoded, Förster resonance energy transfer (FRET) biosensors enable live-cell optical imaging of signaling molecules. Small conformational changes often limit the dynamic range of biosensors that combine fluorescent proteins (FPs) and sensing domains into a single polypeptide. To address this, we developed FRET and lanthanide-based FRET (LRET) biosensors of Rac1 activation with two key features that enhance sensitivity and dynamic range.

Lanthanide-based resonance energy transfer biosensors for live-cell applications.

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enable sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living mammalian cells. LRET biosensors are polypeptides that consist of an alpha-helical linker sequence sandwiched between a lanthanide complex-binding domain and a fluorescent protein (FP) with two interacting domains residing at each terminus. Interaction between the terminal affinity domains brings the lanthanide complex and FP in close proximity such that lanthanide-to-FP, LRET-sensitized emission is increased.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1-92) and HDM2 (1-128).

Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment.

Time-Gated Luminescence Detection of Enzymatically Produced Hydrogen Sulfide: Design, Synthesis, and Application of a Lanthanide-Based Probe.

Hydrogen sulfide (HS) is now recognized as an important gaseous transmitter that is involved in a variety of biological processes. Here, we report the design and synthesis of a luminescent lanthanide biosensor for HS, LP2-Cu(II)-Ln(III), a heterobinuclear metal complex that uses Cu(II) decomplexation to control millisecond-scale-lifetime-Tb(III)- or Eu(III)-emission intensity. LP2-Cu(II)-Ln(III) responded rapidly, selectively, and with high sensitivity to aqueous HS.

Efficient route to pre-organized and linear polyaminopolycarboxylates: Cy-TTHA, Cy-DTPA and mono/di- reactive, -butyl protected TTHA/Cy-TTHA.

Pre-organized polyaminopolycarboxylate chelators Cy-TTHA and Cy-DTPA were synthesized via modular five-step syntheses from commercially available starting materials in ~ 62% and 47% overall yields, respectively. Furthermore, strategies are reported for the efficient preparation of mono- and di-reactive, -butyl-protected TTHA/Cy-TTHA to selectively functionalize central chelators' carboxylic acids.

Time Gated Luminescence Imaging of Immunolabeled Human Tissues.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded tissues is a powerful tool for investigating proteomic profiles and diagnosing disease. However, conventional immunofluorescence with organic dyes is limited in the number of colors that can be simultaneously visualized, is made less sensitive by tissue autofluorescence background, and is usually incompatible with commonly used hematoxylin and eosin staining. Herein, we demonstrate the comparative advantages of using time-gated luminescence microscopy in combination with an emissive Tb(III) complex, Lumi4-Tb, for tissue imaging in terms of sensitivity, multiplexing potential, and compatibility with common immunohistochemistry protocols.

Detection of Hypoxia using a Ratiometric Quantum Dot-based Oxygen Sensor.

A quantum-dot based ratiometric fluorescent oxygen probe for the detection of hypoxia in live cells is reported. The system is comprised of a water-soluble near-infrared emissive quantum dot conjugated to perylene dye. The response to the oxygen concentration is investigated using enzymatic oxygen scavenging in water, while studies were performed with HeLa cells incubated under varying O levels.

Time-Gated Detection of Cystathionine γ-Lyase Activity and Inhibition with a Selective, Luminogenic Hydrogen Sulfide Sensor.

Herein, we report the design, synthesis, and characterization of a lanthanide complex-based probe for the time-gated luminescence detection of hydrogen sulfide (H S) in aqueous media. The probe's unique sensing mechanism relies on the selective reduction of azide to amine by sulfide, followed by intramolecular cyclization to form a quinolinone. The quinolinone is a sensitizer that absorbs near-UV light and transfers excitation energy to coordinated Tb or Eu ions to trigger a strong "turn-on" luminescence response with ms-scale lifetimes characteristic of lanthanide complexes.

Brightly Luminescent and Kinetically Inert Lanthanide Bioprobes Based on Linear and Preorganized Chelators.

The synthesis, photophysical properties, and kinetic stability of a series of water-soluble, highly emissive Tb(III) and Eu(III) complexes featuring triethylenetetraamine hexaacetic acid (TTHA) and cyclohexyl triethylenetetraamine hexaacetic acid (cyTTHA) chelator scaffolds and carbostyril sensitizers are reported. The unique and modular design of the chelators gives rise to striking quantum yields of emission in aqueous solutions (up to 54%) as well as the characteristic lanthanides' photophysical properties (long excited-state lifetimes, large effective Stokes shifts, and narrow emission peaks). Furthermore, the preorganized chelators (L3, L4, and L6) bind metal within minutes at ambient temperature yet exhibit substantial resistance to transchelation in the presence of a challenge solution (EDTA, 1 mM).

Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging.

Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging.

Ratiometric QD-FRET Sensing of Aqueous H2S in Vitro.

We report a platform for the ratiometric fluorescent sensing of endogenously generated gaseous transmitter H2S in its aqueous form (bisulfide or hydrogen sulfide anion) based on the alteration of Förster resonance energy transfer from an emissive semiconductor quantum dot (QD) donor to a dithiol-linked organic dye acceptor. The disulfide bridge between the two chromophores is cleaved upon exposure to bisulfide, resulting in termination of FRET as the dye diffuses away from the QD. This results in enhanced QD emission and dye quenching.

Evaluating the performance of time-gated live-cell microscopy with lanthanide probes.

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts.

Cytoplasmic delivery and selective, multicomponent labeling with oligoarginine-linked protein tags.

Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells' plasma membranes are impermeable to many useful probes and biolabels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels.

How to build a time-gated luminescence microscope.

The sensitivity of filter-based fluorescence microscopy techniques is limited by autofluorescence background. Time-gated detection is a practical way to suppress autofluorescence, enabling higher contrast and improved sensitivity. In the past few years, three groups of authors have demonstrated independent approaches to build robust versions of time-gated luminescence microscopes.

Lanthanide-based imaging of protein-protein interactions in live cells.

In order to deduce the molecular mechanisms of biological function, it is necessary to monitor changes in the subcellular location, activation, and interaction of proteins within living cells in real time. Förster resonance energy-transfer (FRET)-based biosensors that incorporate genetically encoded, fluorescent proteins permit high spatial resolution imaging of protein-protein interactions or protein conformational dynamics. However, a nonspecific fluorescence background often obscures small FRET signal changes, and intensity-based biosensor measurements require careful interpretation and several control experiments.

Time-gated luminescence microscopy with responsive nonmetal probes for mapping activity of protein kinases in living cells.

A photoluminescence probe ARC-1185, possessing both high affinity towards basophilic protein kinases (PKs) and microsecond-scale luminescence lifetime when associated with a kinase, was used for the mapping of ARC-1185-PK complexes in living cells with time-gated luminescence microscopy.

Cell-penetrating peptides as delivery vehicles for a protein-targeted terbium complex.

Release after transmission: Arginine-rich, cell-penetrating peptides (CPPs) mediate cytoplasmic delivery of trimethoprim (TMP)-terbium complex conjugates and selective, intracellular labeling of E. coli dihydrofolate reductase (eDHFR) fusion proteins. A disulfide bond linking CPP and cargo is reduced following uptake.

Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells.

Lanthanide-based or luminescence resonance energy transfer (LRET) microscopy can be used to sensitively image interactions between reporter-labeled proteins in living mammalian cells. With LRET, luminescent lanthanide complexes are used as donors, conventional fluorophores are used as acceptors, and donor-sensitized acceptor emission occurs at time scales that reflect the long (~ms) lanthanide emission lifetime. These long-lived signals can be separated from short-lifetime (~ns) sample autofluorescence and directly excited acceptor fluorescence by using pulsed light to excite the specimen and by implementing a short delay (>100 ns) before detection, thereby increasing measurement sensitivity.

An adaptable luminescence resonance energy transfer assay for measuring and screening protein-protein interactions and their inhibition.

Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays.

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates.

Efficient functionalization of aqueous CdSe/ZnS nanocrystals using small-molecule chemical activators.

Small molecular reagents that can efficiently functionalize water soluble CdSe/ZnS nanocrystals (NCs) are reported. These reagents do not cause quenching or precipitation of NCs as seen with commercially available activators. The results demonstrate that controlling the electrostatic character of the materials is critical in the design of functionalization schemes.

Time-resolved microscopy for imaging lanthanide luminescence in living cells.

Time-resolved luminescence (TRL) microscopy can image signals from lanthanide coordination complexes or other probes with long emission lifetimes, thereby eliminating short-lifetime (<100 ns) autofluorescence background from biological specimens. However, lanthanide complexes emit far fewer photons per unit time than conventional fluorescent probes, making it difficult to rapidly acquire high quality images at probe concentrations that are relevant to live cell experiments. This article describes the development and characterization of a TRL microscope that employs a light-emitting diode (LED, λ(em) = 365 nm) for pulsed epi-illumination and an intensified charge-coupled device (ICCD) camera for gated, widefield detection.

Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells.

Förster resonance energy transfer (FRET) with fluorescent proteins permits high spatial resolution imaging of protein-protein interactions in living cells. However, substantial non-FRET fluorescence background can obscure small FRET signals, making many potential interactions unobservable by conventional FRET techniques. Here we demonstrate time-resolved microscopy of luminescence resonance energy transfer (LRET) for live-cell imaging of protein-protein interactions.