Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival.

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumour formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signalling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signalling, and that drugs designed to target this site may be potent anti-cancer agents.

Plasmon-controlled fluorescence towards high-sensitivity optical sensing.

Fluorescence spectroscopy is widely used in chemical and biological research. Until recently most of the fluorescence experiments have been performed in the far-field regime. By far-field we imply at least several wavelengths from the fluorescent probe molecule. In recent years there has been growing interest in the interactions of fluorophores with metallic surfaces or particles. Near-field interactions are those occurring within a wavelength distance of an excited fluorophore. The spectral properties of fluorophores can dramatically be altered by near-field interactions with the electron clouds present in metals. These interactions modify the emission in ways not seen in classical fluorescence experiments. Fluorophores in the excited state can create plasmons that radiate into the far-field and fluorophores in the ground state can interact with and be excited by surface plasmons. These reciprocal interactions suggest that the novel optical absorption and scattering properties of metallic nanostructures can be used to control the decay rates, location, and direction of fluorophore emission. We refer to these phenomena as plasmon-controlled fluorescence (PCF). An overview of the recent work on metal-fluorophore interactions is presented. Recent research combining plasmonics and fluorescence suggest that PCF could lead to new classes of experimental procedures, novel probes, bioassays, and devices.

Enhanced fluorescence from periodic arrays of silver nanoparticles.

Electron beam lithography was used to fabricate silver nanoparticle arrays and study the effects of geometrical properties of particles on metal-enhanced fluorescence. Nanoparticle size, shape, interparticle spacing, and nominal thickness were varied in a combinatorial pattern for investigation of the particle plasmon resonance effect on enhancement of fluorescence from three different fluorophores; Fluorescein, Cy3, and Cy5. A specific geometric property for optimal enhancement from each fluorophore was determined. For interparticle spacings greater or equal to 270 nm, the enhancement matched what is expected for a single-particle model. For those particles smaller than 210 nm, the enhancement was lower than for the larger spacing in the range studied. Triangular-shaped particles gave similar enhancement to those of square-shaped particles. This combinatorial pattern by e-beam lithography was found to be useful for studying how individual parameters enhance the fluorescence that are important for rational design of enhanced fluorescence sensors.

Synthesis, spectral properties, and detection limits of reactive squaraine dyes, a new class of diode laser compatible fluorescent protein labels.

We describe the synthesis and spectral characterization of two reactive long-wavelength fluorescence labels (Sq635-m and Sq635-b), having either one or two N-hydroxysuccinimidyl esters. Both are squaraine derivatives and consist of a cyanine-type chromophore and a central squarate bridge. To improve water solubility, we introduced two sulfonic acid groups into the heterocyclic ring systems, and for covalent attachment to proteins, a reactive N-hydroxy-succinimide ester (NHS ester) was synthesized. The squaraine markers exhibit low quantum yields in water (phi = 0.15) and high quantum yields (phi = 0.6-0.7) when bound to proteins. The absorption maxima at 635 nm in water and at approximately 645 nm when bound to proteins allow excitation with commercially available diode lasers. The detection limit of a representative squaraine dye in blood was estimated to be half that of a commonly used fluorophore.

Lifetime-based pH sensors: indicators for acidic environments.

We characterized the pH-dependent intensity decays of three fluorophores, Oregon green 514 carboxylic acid, Cl-NERF, and DM-NERF, using frequency-domain fluorometry, with the objective of identifying lifetime-based sensors for low pH values. These three probes were originally designed as dual excitation wavelength-ratiometric probes, with high photostability and high quantum yields in aqueous solutions. We found that their fluorescence intensity decays were strongly dependent on pH. Moreover, global intensity decays analysis reveals that these probes have double exponential intensity decays at intermediate pH values and that the decay time amplitudes are greatly dependent on pH. The longer lifetime components originated from the unprotonated forms and the shorter components from the protonated forms. Both forms can emit fluorescence at intermediate pH values. The apparent pKa values were also determined from the titration curves of phase angles and modulations versus pH for the purpose of pH sensing. The apparent pKa values range from pH 3 to 5, a range where lifetime-based sensors are not presently reported. Since these probes show low pKa values and display substantial phase and modulation changes with pH, they are suitable as lifetime-based pH sensors to monitor the pH changes in acidic environments. One potential application of these probes is to trace the pH in different cellular compartments.

Spatially localized ballistic two-photon excitation in scattering media.

We describe spatially localized two-photon excitation in scattering media. Using femtosecond pulses at 770 nm from a Ti: Sapphire laser, we were able to excite fluorophores in capillary tubes under up to 1.5 mm of 0.5% intralipid. Displacement of the laser beam relative to the embedded samples indicates that highly localized excitation was possible with two-photon excitation, whereas one-photon excitation resulted in loss of spatial resolution due to excitation by the diffusely scattered photons. These results indicate that two-photon excitation in the scattering solution is due only to the ballistic photons, a result confirmed by frequency-domain time-resolved measurements. Selective excitation of adjacent embedded samples was found possible for two but not one-photon excitation.

Long-lifetime Ru(II) complexes for the measurement of high molecular weight protein hydrodynamics.

We describe the synthesis and characterization of two asymmetrical ruthenium(II) complexes, [Ru(dpp)2(dcbpy)]2+ and [Ru(dpp)2(mcbpy)]2+, as well as the water soluble sulfonated derivatives [Ru(dpp(SO3Na)2)2(dcbpy)]2+ and [Ru(dpp(SO3Na)2)2(mcbpy)]2+ (dpp is 4,7-diphenyl-1,10-phenanthroline, dcbpy is 4,4'-dicarboxylic acid-2,2'-bipyridine, mcbpy is 4-methyl,4'-carboxylic acid-2,2'-bipyridine, and dpp(SO3Na)2 is the disulfonated derivative of dpp) as probes for the measurement of the rotational motions of proteins. The spectral (absorption, emission, and anisotropy) and photophysical (time-resolved intensity and anisotropy decays) properties of these metal-ligand complexes were determined in solution, in both the presence and absence of human serum albumin (HSA). These complexes display lifetimes ranging from 345 ns to 3.8 microseconds in deoxygenated aqueous solutions under a variety of conditions. The carboxylic acid groups on these complexes were activated to form N-hydroxysuccinimide (NHS) esters which were used to covalently lable HSA, and were characterized spectroscopically in the same manner as above. Time-resolved anisotropy measurements were performed to demonstrate the utility of these complexes in measuring long rotational correlation times of bioconjugates between HSA and antibody to HSA. The potential usefulness of these probes in fluorescence polarization immunoassays was demonstrated by an association assay of the Ru(II)-labeled HSA with polyclonal antibody.

Synthesis and spectral characterization of a thiol-reactive long-lifetime Ru(II) complex.

We report the synthesis and spectral properties of a long-lifetime luminescent Ru complex containing a sulfhydryl-reactive maleimide group, [Ru (2,2'-bipyridine)2(1, 10-phenanthroline-5-maleimide)](PF6)2. [Ru(bpy)2(phen-mi)]2+ was covalently linked to human serum albumin, immunoglobulin G, and beta-galactosidase. The lifetimes for probe bound to proteins were near 1.1 micros. In the absence of rotational motions, the probe displayed an anisotropy near 0.17 for excitation near 475 nm. Anisotropy decay data were used to determine rotational correlation times of the proteins, which showed local probe motions in addition to overall rotational diffusion. This long-lifetime sulfhydryl-reactive probe can be used to recover microsecond rotational motions and/or domain motions of proteins and/or macromolecular complexes.

Sodium Green as a potential probe for intracellular sodium imaging based on fluorescence lifetime.

We characterized the use of the fluorescent probe Sodium Green for measurements of intracellular free sodium using frequency-domain, phase-modulation fluorometry. The intensity decays were found to be strongly Na+ dependent, with mean lifetime increasing from 1.13 ns in the absence of Na+ to 2.39 ns in the presence of 140 mM Na+. Detailed analysis of the intensity decays in the presence of Na+ and K+ in the concentration range from 0 to 500 mM is provided. Sodium sensing using data measured at a single modulation frequency is described. Phase and modulation data showed high sensitivity to Na+ and substantially lower sensitivity to K+. Additionally, exposure of Sodium Green to intense illumination indicated that Sodium Green is much more photostable than its precursor, fluorescein. These results indicate that lifetime-based measurements with Sodium Green can be used for imaging of intracellular free [Na+] in the range from about 0.5 to 50 mM with high accuracy.

Lifetime-based sensing of glucose using energy transfer with a long lifetime donor.

We describe an optical assay for glucose based on the luminescence decay time of a long lifetime metal-ligand complex. Concanavalin A was covalently labeled with Ruthenium metal-ligand complex (RuCon A) which served as the donor. The acceptor was malachite green which was covalently linked to insulin. The malachite green insulin was also covalently labeled with maltose (MIMG) to provide binding affinity to RuCon A. Binding of RuCon A to MIMG resulted in a decreased intensity and decay time of RuCon A. Glucose was detected by competitive displacement of MIMG from RuCon A, resulting in increased intensity and decay time. This glucose assay has several favorable features. The long lifetime of RuCon A allows phase-modulation decay time measurements using an amplitude-modulated bluelight-emitting diode as the light source. Reversibility of the assay can be controlled by the extent of sugar labeling of the insulin. Finally, the glucose-sensitive range can be adjusted by selection of the sugar structure and extent of labeling of the insulin.

Fluorescence study of neurohypophyseal hormones and their analogues. Distance distributions in a series of arginine-vasopressin analogues.

Analogues of arginine-vasopressin (AVP) in which substitution of the proline residue in position 7 (by either sarcosine or N-methylalanine) combined with replacement of the cysteine residue in position 1 were the subject of a fluorescence and molecular mechanics study. We obtained two groups of analogues: selective antidiuretic agonists (cysteine or beta-mercaptopropionic acid in position 1) and pressor and uterotonic antagonists (deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid in position 1). Using frequency-domain measurements of fluorescence resonance energy transfer (FRET) we estimated the distance distribution between the phenolic ring of Tyr2 and the disulphide bridge Cys1-Cys6. We also analyzed acrylamide quenching of tyrosyl fluorescence to determine the exposure of the tyrosyl ring to the solvent. Results from fluorescence experiments were compared with those from Monte Carlo simulation (ECEPP/3 force-field).

Synthesis and luminescence spectral characterization of long-lifetime lipid metal-ligand probes.

We synthesized phospholipid analogues of phosphatidyl ethanolamine which contains a ruthenium metal-ligand complex (MLC) covalently bound to the amino group. Two analogues were synthesized, containing either one (Ru-PE) or two (Ru-PE2) lipid molecules covalently linked to the MLC by the amino group of the lipid. These MLC-lipid probes display intensity decay times from 682 to 357 ns, depending on temperature. Importantly, the luminescence MLC groups display polarized emission, enabling their use for studies of membrane dynamics. The long intensity decay times allowed measurement of the overall rotation correlation time of lipid vesicles to several microseconds. The spectral properties of the model membranes containing Ru-PE or Ru-PE2 were independent of the probe-to-lipid molar ratio from 1:20 to 1:100, suggesting minimal tendency for probe-probe interactions. These MLC-lipid probes can be expected to have numerous applications in studies of membrane dynamics on the microsecond timescale.

A long-lived, highly luminescent Re(I) metal-ligand complex as a biomolecular probe.

A highly luminescent rhenium (I) metal-ligand complex [Re(bcp)(CO)3(4-COOHPy)](ClO4), where bcp is 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been synthesized and characterized. High quantum yields (> 0.5) and long excited-state lifetimes (0.3-10 micronseconds) in fluid solutions at room temperature were found for this complex, with remarkable emission sensitivity to microenvironment. This compound also displays highly polarized emission with a maximum anisotropy near 0.3 in the absence of rotational diffusion. This Re complex was conjugated to several biomolecules, including the proteins human serum albumin and bovine immunoglobulin G, as well as an amine-containing lipid. When bound to a protein or lipid, the decay time is near 3 microseconds and the quantum yield is approximately 0.12 in aqueous oxygenated solution at room temperature. This compound's unique spectral properties along with its conjugatability allowed us to utilize it as biomolecular probe in a variety of environments.

Synthesis and evaluation of Ru-complexes as anisotropy probes for protein hydrodynamics and immunoassays of high-molecular-weight antigens.

We investigated three unsymmetrical Ru-complexes, namely [Ru(bpy)2 (phen-ITC)]2+, [Ru(bpy)2(dcbpy)] and [Ru(bpy)2(mcbpy)]+ for use as probes for rotational diffusion and in immunoassays of high-molecular-weight antigens. For this purpose we synthesized reactive forms of these metal-ligand complexes and conjugated them to human serum albumin (HSA). The maximal anisotropies (r0) for the HSA-bound forms in frozen solution are 0.23, 0.17 and 0.14 for the (dcbpy), (mcbpy) and (phen-ITC) derivatives, respectively. The activated Ru metal-ligand complexes have either one or two NHS-esters or an isothiocyanate group as the reactive moiety. The usefulness of these complexes in immunoassays was determined by titration of the labeled HSA with polyclonal anti-HSA. The highest steady state anisotropy (r) values (0.190) were observed for the [Ru(bpy)2(dcbpy)]-labeled HSA on titration with polyclonal antibody. However, a relative increase in the steady state anisotropy (r/r0) on titration with polyclonal antibody was found for the phen-ITC probe (96%), as compared to the dcbpy (83%) or mcbpy (79%) derivatives. These findings were confirmed by time-resolved frequency-domain measurements. In particular the higher mean correlation times calculated for the phen-ITC derivative suggests reduced local probe motion for this probe when bound to HSA as compared to the (mcbpy) and (dcbpy) conjugates.

Fluorescence polarization immunoassay of a high-molecular-weight antigen using a long wavelength-absorbing and laser diode-excitable metal-ligand complex.

We describe a fluorescence polarization immunoassay (FPI) based on an osmium metal-ligand complex, Os(bpy)2(dcbpy). The Os complex was selected for its long wavelength absorption, which allows excitation with wavelengths up to 720 nm. At these wavelengths tissue absorbance and autofluorescence are minimal, and excitation can be accomplished with LEDs or laser diodes. The Os complex displays a lifetime of 19 ns and a high initial anisotropy at two excitation wavelengths, 505 and 690 nm. A reactive NHS-ester of Os- (bpy)2(dcbpy) was synthesized containing a NHS ester, and used to label the human serum albumin (HSA) or anti-HSA. The FPIs of HSA were performed with monoclonal and polyclonal antibodies using excitation at 505 or 685 nm. The results showed the potential of the Os-ligand complex to be used as a long excitation wavelength FPI probe for potential use in homogeneous immunoassays with simple excitation sources.

Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer.

We have examined the fluorescence intensity decays of oxytocin and [Arg8]-vasopressin resulting from the single tyrosyl residue in each peptide, and the intensity decay of the Asu1,6-analogues in which the disulfide bridge is substituted by a CH2-CH2 bridge. Viscosity-dependent steady state and intensity decay measurements indicated that fluorescence resonance energy transfer (FRET) from tyrosyl phenol to the disulfide bridge is responsible for the decrease in fluorescence relative to the Asu-analogues. The frequency-domain phase and modulation data for the tyrosyl donor were interpreted in terms of fluorescence resonance energy transfer (FRET) to the weakly absorbing disulfide bridge and a distribution of donor-to-acceptor distances. Energy transfer efficiencies were determined from both time-resolved and steady-state measurements. Fitting the frequency-domain phase and modulation data to a Gaussian distance distribution indicated that the average inter-chromophoric distance (Rav) is similar in both compounds, Rav = 7.94 A for oxytocin and Rav = 8.00 A for vasopressin. However, the width of the distance distribution is narrower for vasopression (hw = 2.80 A) than for oxytocin (hw = 3.58 A), which is consistent with restriction of the tyrosine phenol motion due to its stacking wih the Phe3 side chain of vasopressin. Finally, the recovered distance distribution functions are compared with histograms describing the distance between the chromophores during the course of long, in vacuo, molecular dynamics runs using the computer program CHARMm and the QUANTA 3.0 parameters.

Three-photon induced fluorescence of the calcium probe Indo-1.

We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.