RESUMEN
Controlled light-mediated delivery of biological analytes can enable the investigation of highly reactivity molecules within living systems. As many biological effects are concentration dependent, it is critical to determine the location, time, and quantity of analyte donation. In this work, we have developed the first photoactivatable donor for formaldehyde (FA). Our optimized photoactivatable donor, photoFAD-3, is equipped with a fluorescence readout that enables monitoring of FA release with a concomitant 139-fold fluorescence enhancement. Tuning of photostability and cellular retention enabled quantification of intracellular FA release through cell lysate calibration. Application of photoFAD-3 uncovered the concentration range necessary for arresting wound healing in live cells. This marks the first report where a photoactivatable donor for any analyte has been used to quantify intracellular release.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Formaldehído/farmacología , Fluorescencia , Colorantes Fluorescentes/química , Formaldehído/química , Células HEK293 , Células HeLa , HumanosRESUMEN
Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.
Asunto(s)
Cobre/farmacología , AMP Cíclico/metabolismo , Lipólisis/efectos de los fármacos , Inhibidores de Fosfodiesterasa 3/farmacología , Células 3T3-L1 , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Relación Estructura-ActividadRESUMEN
DNA aptamers are a powerful class of molecules for sensing targets, but have been limited when applied to imaging in living animals because most aptamer probes are fluorescence-based, which limits imaging penetration depth. Photoacoustic (PA) imaging emerged as an alternative to MRI and X-ray tomography in biomedical imaging, due to its ability to afford high-resolution images at depths in the cm range. Despite its promise, PA imaging is limited by a lack of strategies to design selective and activatable probes for targets. To overcome this limitation, we report design and demonstration of PA probes based on DNA aptamers that can hybridize to DNA strands conjugated to a near-infrared fluorophore/quencher pair (IRDye 800CW/IRDye QC-1) with efficient contact quenching. Binding of the target triggered a release of the DNA strand with the quencher and thus relief of the contact quenching, resulting in a change of the PA signal ratio at 780/725 nm. Using thrombin as a model, a relationship was established between the thrombin concentrations and the PA ratio, with a dynamic range of 0-1000 nM and a limit of detection of 112 nM. Finally, in vivo PA imaging studies showed that the PA ratio increased significantly 45 min after injection of thrombin but not with injection of PBS as a vehicle control, demonstrating the first aptamer-based activatable PA probe for advanced molecular imaging in living mice. Since in vitro selection can obtain aptamers selective for many targets, the design demonstrated can be applied for PA imaging of a number of targets.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , ADN/metabolismo , Imagen Molecular/métodos , Técnicas Fotoacústicas/métodos , Animales , Colorantes Fluorescentes/metabolismo , Rayos Infrarrojos , Ratones , Sondas Moleculares/metabolismo , Trombina/metabolismoRESUMEN
Photoacoustic tomography has emerged as a promising alternative to MRI and X-ray scans in the clinical setting due to its ability to afford high-resolution images at depths in the cm range. However, its utility has not been established in the basic research arena owing to a lack of analyte-specific photoacoustic probes. To this end, we have developed acoustogenic probes for copper(II)-1 and -2 (APC-1 and APC-2, a water-soluble congener) for the chemoselective visualization of Cu(II), a metal ion which plays a crucial role in chronic neurological disorders such as Alzheimer's disease. To detect Cu(II), we have equipped both APCs with a 2-picolinic ester sensing module that is readily hydrolyzed in the presence of Cu(II) but not by other divalent metal ions. Additionally, we designed APC-1 and APC-2 explicitly for ratiometric photoacoustic imaging by using an aza-BODIPY dye scaffold exhibiting two spectrally resolved NIR absorbance bands which correspond to the 2-picolinic ester capped and uncapped phenoxide forms. The normalized ratiometric turn-on responses for APC-1 and APC-2 were 89- and 101-fold, respectively.
Asunto(s)
Acústica , Cobre/química , Sondas MolecularesRESUMEN
SWIR dyes offer many advantages over their more common NIR congeners; however, the available options are limited. New SWIR imaging agents can be accessed by remodeling existing NIR molecules (i.e., hemicyanines (HDs)). In this study, we synthesized SWIR-HD, a modified HD featuring dimethylsilicon and benzo[cd]indolium groups that are designed to red-shift the absorbance and emission to 988 and 1126 nm, respectively. SWIR-HD was employed to visualize the liver and tumors via multimodal SWIR imaging.