Photoactivatable drugs for nicotinic optopharmacology.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Fluorogenic cephalosporin substrates for ß-lactamase TEM-1.

Cephalosporin was used to synthesize soluble and precipitating fluorogenic ß-lactam substrates that demonstrated differential catalytic hydrolysis by three different subtypes of ß-lactamase: TEM-1 (class A), p99 (class C), and a Bacillus cereus enzyme sold by Genzyme (class B). The most successful soluble substrate contained difluorofluorescein (Oregon Green 488) ligated to two cephalosporin moieties that, therefore, required two turnovers to produce the fluorescent Oregon Green 488 leaving group. The bis-cephalosporin modification was required so that the final reaction product was the Oregon Green 488 carboxylic acid rather than a less bright phenolic adduct of the dye. Hydrolysis in pH 5.5 Mes and pH 7.2 phosphate-buffered saline (PBS) buffers was similar, but in pH 8.0 Tris the hydrolysis rate nearly doubled. Activity of the ß-lactamases on the various substrates was shown to depend highly on the linker between the cephalosporin and the fluorophore, with an allyl linker promoting faster turnover than a phenol ether linker. Measured K(m) values for dichlorofluorescein and difluorofluorescein cephalosporin substrates were approximately the same as K(m) values for penicillin G and ampicillin found in the literature (~30-40µM).

Low-affinity Ca2+ indicators compared in measurements of skeletal muscle Ca2+ transients.

The low-affinity fluorescent Ca(2+) indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca(2+) transient (Delta[Ca(2+)]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, furaptra (previously shown to rapidly track Delta[Ca(2+)]). In response to an action potential, the full duration at half-maximum of the indicator's fluorescence change (DeltaF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with furaptra; thus, these indicators do not track Delta[Ca(2+)] with kinetic fidelity. In contrast, the DeltaF time course of Mag-fluo-4 was identical to furaptra's; thus, Mag-fluo-4 also yields reliable kinetic information about Delta[Ca(2+)]. Mag-fluo-4's DeltaF has a larger signal/noise ratio than furaptra's (for similar indicator concentrations), and should thus be more useful for tracking Delta[Ca(2+)] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg(2+), the amplitude of the Mag-fluo-4 signal, and its calibration in Delta[Ca(2+)] units, is likely to be more sensitive to variations in [Mg(2+)] than furaptra's.

Fluorescent labeling of proteins in living cells using the FKBP12 (F36V) tag.

Over the past decade live cell imaging has become a key technology to monitor and understand the dynamic behavior of proteins in the physiological context of living cells. The visualization of a protein of interest is most commonly achieved by genetically fusing it to green fluorescent protein (GFP) or one of it variants. Considerable effort has been made to develop alternative methods of protein labeling to overcome the intrinsic limitations of fluorescent proteins. In this report we show the optimization of a live cell labeling technology based on the use of a mutant form of FKBP12 (FKBP12(F36V)) in combination with a synthetic high affinity ligand (SLF') that specifically binds to this mutant. It had been previously shown that the use of a fluorescein-conjugated form of SLF' (5'-fluorescein-SLF') allowed the labeling of proteins genetically fused to FKBP-F36V in living cells. Here we describe the identification of novel fluorescent SLF'dye conjugates that allow specific labeling of FKBP12(F36V) fusion proteins in living cells. To further increase the versatility of this technology we developed a number of technical improvements. We implemented the use of pluronics during the labeling process to facilitate the uptake of the SLF'-dye conjugates and the use suppression dyes to reduce background signal. Furthermore, the time and dose dependency of labeling was investigated in order to determine optimal labeling conditions. Finally, the specificity of the FKBP12(F36V) labeling technology was extensively validated by morphological analysis using a diverse set of FKBP12(F36V) fusions proteins. In addition we show a number of different application examples, such as translocation assays, the generation of biosensors, and multiplex labeling in combination with different labeling technologies, such as FlAsH or GFP. In summary we show that the FKBP12(F36V)/SLF' labeling technology has a broad range of applications and should prove useful for the study of protein function in living cells.

The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium.

There has been some dispute in the literature as to the sensitivity of the zinc indicator FluoZin-3 to calcium, with suggestions that physiological levels of calcium and magnesium effectively occlude the response of the probe to zinc. In this communication we demonstrate that calcium concentrations as high as 10 mM do not prevent FluoZin-3 from detecting zinc elevations as low as 100 pM. Moreover, the inclusion of a few microM Ca-EDTA does not prevent FluoZin-3 from responding to increases in zinc concentration but does extend the dynamic range of the probe by reducing contaminating zinc levels and allowing the probe to respond to multiple zinc additions. In addition, we have derived a mathematical model to account for the kinetics of FluoZin-3 response to zinc in the presence of an additional zinc and calcium chelator.

Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.

Water-soluble poly(2,7-dibenzosilole) as an ultra-bright fluorescent label for antibody-based flow cytometry.

A series of novel water-soluble PEGylated dibenzosilole-based conjugated polymers were prepared as ultra-bright fluorescent labels for biomolecules. Due to their superior solubility and brightness, antibody conjugates labeled with functionalized polymers showed significantly enhanced signal and sensitivity relative to traditional fluorophores in functional flow cytometry applications.

Control of DNA hybridization with photocleavable adducts.

Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence-based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14-16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity.

A new mitochondrial fluorescent zinc sensor.

A novel cationic fluorescent zinc (Zn2+) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+]m).