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Hailin Wang - One of the best experts on this subject based on the ideXlab platform.
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nucleic acid aptamers improving Fluorescence Anisotropy and Fluorescence polarization assays for small molecules
Trends in Analytical Chemistry, 2019Co-Authors: Qiang Zhao, Jagdeesh S Uppal, Hanyong Peng, Hailin Wang, Chris X LeAbstract:Abstract Affinity probes, such as nucleic acid aptamers, have been combined with Fluorescence Anisotropy (FA)/Fluorescence polarization (FP) technology for the development of a diverse range of assays. Formation of a complex between a small fluorescent molecule and its binding partner usually increases the overall size of the fluorescent molecule and decreases its rate of rotation, resulting in increases in Fluorescence Anisotropy/polarization. Structure-switching of the fluorescently labeled aptamers arising from target binding can also affect molecular volume, local rotation of the fluorophore, and/or Fluorescence lifetime, causing changes in Anisotropy/polarization. Incorporation of the unique adsorptive properties of single-stranded nucleic acid aptamers on nanomaterials, hybridization of aptamers with complementary sequences, and the amplifiable ability of nucleic acid aptamers have broadened the applications of Fluorescence Anisotropy assays and enhanced their sensitivity. This review focuses on nucleic acid aptamer-based Fluorescence Anisotropy assays for the detection of small molecules, such as therapeutic drugs, environmental contaminants, natural toxins, and metabolites.
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nanoparticles free Fluorescence Anisotropy amplification assay for detection of rna nucleotide cleaving dnazyme activity
Analytical Chemistry, 2015Co-Authors: Dapeng Zhang, Qiang Zhao, Haiqin Rong, Hailin WangAbstract:Fluorescence Anisotropy is a homogeneous, sensitive, ratiometric, and real-time analytical technology. However, it is a great challenge to produce a large Fluorescence Anisotropy change upon the presence of target small molecules without nanoparticles-dependent amplification. This work reports a nanoparticle-free and multiple G-enhanced Fluorescence Anisotropy assay for detection of DNAzyme activity. A Pb2+-dependent GR-5 DNAzyme was used as a model. We hybridized the rA-cleavable substrate strand containing a TMR label at the 5′-end with the DNAzyme strand containing an extended three G bases at the 3′-end. By this design, we demonstrate that both Fluorescence quenching and the enhanced DNAzyme activity contribute to a Pb2+-induced large Fluorescence Anisotropy change (|Δr| = 0.168). The limit of detection for Pb2+ is estimated to be about 100 pM with a dynamic range from 200 pM to 100 nM. The interference from the other nine divalent metal ions of 1000-times excess amount is negligible. Moreover, we sho...
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Aptamer Fluorescence Anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides.
Biosensors & bioelectronics, 2015Co-Authors: Qiang Zhao, Hailin WangAbstract:We previously reported a Fluorescence Anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer Fluorescence Anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMR-labeled aptamers enabled direct FA sensing ATP with a detection limit of 1 µM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets.
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Identification of allosteric nucleotide sites of tetramethylrhodamine-labeled aptamer for noncompetitive aptamer-based Fluorescence Anisotropy detection of a small molecule, ochratoxin A.
Analytical chemistry, 2013Co-Authors: Qiang Zhao, Hailin WangAbstract:Aptamer-based Fluorescence Anisotropy (FA) assay combines the advantages of affinity aptamers in good stability, easy generation, and facile labeling and the benefits of FA in homogeneous analysis, such as robustness, simplicity, and high reproducibility. By using a fluorophore-labeled aptamer, FA detection of a small molecule is not as easy as detection of protein because the binding of a small molecule cannot cause significant increase of molecular weight of the dye-labeled aptamer. The intramolecular interaction between labeled tetramethylrhodamine (TMR) and DNA aptamer bases dramatically affects the local rotation and FA of TMR. This intramolecular interaction can be altered by aptamer conformation change upon target binding, leading to a significant change of FA of TMR. Taking this unique feature of a TMR-labeled aptamer, we described a noncompetitive aptamer-based Fluorescence Anisotropy assay for detection of small molecules by using ochratoxin A (OTA) as a model. We successfully identified the spe...
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screening interaction between ochratoxin a and aptamers by Fluorescence Anisotropy approach
Analytical and Bioanalytical Chemistry, 2013Co-Authors: Xia Geng, Dapeng Zhang, Hailin Wang, Qiang ZhaoAbstract:By taking advantage of the intrinsic Fluorescence of ochratoxin A (OTA), we present a Fluorescence Anisotropy approach for rapid analysis of the interactions between OTA and aptamers. The specific binding of OTA with a 36-mer aptamer can induce increased Fluorescence Anisotropy (FA) of OTA as the result of the freedom restriction of OTA and the increase of molecular volume, and the maximum FA change is about 0.160. This FA approach enables an easy way to investigate the effects of buffer compositions like metal ions on the affinity binding. FA analysis shows the interaction between OTA and aptamer is greatly enhanced by the simultaneous presence of Ca2+ and Na+, while the binding affinity of aptamer decreases more than 18-fold when only Ca2+ exists, and the binding is completely lost when Ca2+ is absent. Crucial region of the aptamer for binding can be mapped through FA analysis and aptamer mutation. The demonstrated FA approach maintains the advantages of FA in simplicity, rapidity, and robustness. This investigation will help the development of aptamer-based assays for OTA detection in optimizing the binding conditions, modification of aptamers, and rational design.
Cheng Zhi Huang - One of the best experts on this subject based on the ideXlab platform.
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exonuclease iii assisted graphene oxide amplified Fluorescence Anisotropy strategy for ricin detection
Biosensors and Bioelectronics, 2016Co-Authors: Xue Xiao, Cheng Zhi Huang, Jing Tao, Hong Zhi Zhang, Shu Jun ZhenAbstract:Abstract Graphene oxide (GO) is an excellent Fluorescence Anisotropy (FA) amplifier. However, in the conventional GO amplified FA strategy, one target can only induce the FA change of one fluorophore on probe, which limits the detection sensitivity. Herein, we developed an exonuclease III (Exo III) aided GO amplified FA strategy by using aptamer as an recognition element and ricin B-chain as a proof-of-concept target. The aptamer was hybridized with a blocker sequence and linked onto the surface of magnetic beads (MBs). Upon the addition of ricin B-chain, blocker was released from the surface of MBs and hybridized with the dye-modified probe DNA on the surface of GO through the toehold-mediated strand exchange reaction. The formed blocker–probe DNA duplex triggered the Exo III-assisted cyclic signal amplification by repeating the hybridization and digestion of probe DNA, liberating the fluorophore with several nucleotides (low FA value). Thus, ricin B-chain could be sensitively detected by the significantly decreased FA. The linear range was from 1.0 μg/mL to 13.3 μg/mL and the limit of detection (LOD) was 400 ng/mL. This method improved the sensitivity of FA assay and it could be generalized to any kind of target detection based on the use of an appropriate aptamer.
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a novel graphene oxide amplified Fluorescence Anisotropy assay with improved accuracy and sensitivity
Chemical Communications, 2015Co-Authors: Xue Xiao, Cheng Zhi Huang, Shu Jun ZhenAbstract:In this contribution, a novel and versatile graphene oxide (GO) amplified Fluorescence Anisotropy (FA) strategy with improved accuracy and sensitivity for the detection of a panel of molecules, single-stranded DNA (ssDNA), adenosine and thrombin, has been successfully developed.
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metal organic framework mil 101 enhanced Fluorescence Anisotropy for sensitive detection of dna
RSC Advances, 2014Co-Authors: Jing Fang Guo, Cheng Zhi HuangAbstract:The metal–organic framework (MOF) was first utilized as an amplification platform for Fluorescence Anisotropy assay, which was identified to be effective for highly sensitive detection of DNA.
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a graphene oxide enhanced Fluorescence Anisotropy strategy for dnazyme based assay of metal ions
Chemical Communications, 2013Co-Authors: Yue Liu, Shu Jun Zhen, Cheng Zhi HuangAbstract:Graphene oxide (GO) introduced to enhance the Fluorescence Anisotropy (FA) of fluorogens was identified to be effective for highly sensitive and selective detection of metal ions through an Anisotropy DNAzyme-based strategy.
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graphene signal amplification for sensitive and real time Fluorescence Anisotropy detection of small molecules
Analytical Chemistry, 2013Co-Authors: Changyao Wang, Ronghua Yang, Ying Jiang, Yaping Hu, Jishan Li, Sheng Yang, Yinhui Li, Cheng Zhi HuangAbstract:Fluorescence Anisotropy (FA) is a reliable, sensitive, and robust assay approach for determination of many biological targets. However, it is generally not applicable for the assay of small molecules because their molecular masses are relatively too small to produce observable FA value changes. To address this issue, we report herein the development of a FA signal amplification strategy by employing graphene oxide (GO) as the signal amplifier. Because of the extraordinarily larger volume of GO, the fluorophore exhibits very high polarization when bound to GO. Conversely, low polarization is observed when the fluorophore is dissociated from the GO. As proof-of-principle, the approach was applied to FA detection of adenosine triphosphate (ATP) with a fluorescent aptamer. The aptamer exhibits very high polarization when bound to GO, while the FA is greatly reduced when the aptamer complexes with ATP, which exhibits a maximum signal change of 0.316 and a low detection limit of 100 nM ATP in buffer solution. S...
Dapeng Zhang - One of the best experts on this subject based on the ideXlab platform.
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nanoparticles free Fluorescence Anisotropy amplification assay for detection of rna nucleotide cleaving dnazyme activity
Analytical Chemistry, 2015Co-Authors: Dapeng Zhang, Qiang Zhao, Haiqin Rong, Hailin WangAbstract:Fluorescence Anisotropy is a homogeneous, sensitive, ratiometric, and real-time analytical technology. However, it is a great challenge to produce a large Fluorescence Anisotropy change upon the presence of target small molecules without nanoparticles-dependent amplification. This work reports a nanoparticle-free and multiple G-enhanced Fluorescence Anisotropy assay for detection of DNAzyme activity. A Pb2+-dependent GR-5 DNAzyme was used as a model. We hybridized the rA-cleavable substrate strand containing a TMR label at the 5′-end with the DNAzyme strand containing an extended three G bases at the 3′-end. By this design, we demonstrate that both Fluorescence quenching and the enhanced DNAzyme activity contribute to a Pb2+-induced large Fluorescence Anisotropy change (|Δr| = 0.168). The limit of detection for Pb2+ is estimated to be about 100 pM with a dynamic range from 200 pM to 100 nM. The interference from the other nine divalent metal ions of 1000-times excess amount is negligible. Moreover, we sho...
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screening interaction between ochratoxin a and aptamers by Fluorescence Anisotropy approach
Analytical and Bioanalytical Chemistry, 2013Co-Authors: Xia Geng, Dapeng Zhang, Hailin Wang, Qiang ZhaoAbstract:By taking advantage of the intrinsic Fluorescence of ochratoxin A (OTA), we present a Fluorescence Anisotropy approach for rapid analysis of the interactions between OTA and aptamers. The specific binding of OTA with a 36-mer aptamer can induce increased Fluorescence Anisotropy (FA) of OTA as the result of the freedom restriction of OTA and the increase of molecular volume, and the maximum FA change is about 0.160. This FA approach enables an easy way to investigate the effects of buffer compositions like metal ions on the affinity binding. FA analysis shows the interaction between OTA and aptamer is greatly enhanced by the simultaneous presence of Ca2+ and Na+, while the binding affinity of aptamer decreases more than 18-fold when only Ca2+ exists, and the binding is completely lost when Ca2+ is absent. Crucial region of the aptamer for binding can be mapped through FA analysis and aptamer mutation. The demonstrated FA approach maintains the advantages of FA in simplicity, rapidity, and robustness. This investigation will help the development of aptamer-based assays for OTA detection in optimizing the binding conditions, modification of aptamers, and rational design.
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Fluorescence Anisotropy reduction of allosteric aptamer for sensitive and specific protein signaling
Analytical Chemistry, 2012Co-Authors: Dapeng Zhang, Qiang Zhao, Bailin Zhao, Hailin WangAbstract:Real time protein signaling in a complex medium may provide a promising way for high-throughput protein analysis, but it is largely unmet due to the challenge of signal transduction and the interferences of nonspecific binding and high background. Our recent work indicates that a fluorescent aptamer can display a protein binding-induced reduction of Fluorescence Anisotropy (FA) (Zhang, D.; Lu, M.; Wang, H. J. Am. Chem. Soc.2011, 133, 9188–9191), which is exclusively different from a traditionally simplified concept hinting a molecular size increase-induced FA increase. Inspired by this unexpected observation, we describe a novel FA reduction approach for protein signaling. The feasibility of this approach is demonstrated through the assays of a blood protein human α-thrombin and an oncoprotein human platelet-derived growth factor B-chain (PDGF-BB) using two screened fluorescent aptamers, respectively. By the developed FA reduction method, the spiked human α-thrombin in diluted serum can be detected at the...
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Fluorescence Anisotropy analysis for mapping aptamer protein interaction at the single nucleotide level
Journal of the American Chemical Society, 2011Co-Authors: Dapeng Zhang, Hailin WangAbstract:Structural characterization of aptamer–protein interactions is challenging and limited despite the tremendous applications of aptamers. Here we for the first time report a Fluorescence Anisotropy (FA) approach for mapping the interaction of an aptamer and its protein target at the single nucleotide level. Nine fluorescently labeled aptamers, each conjugated to a single tetramethylrhodamine at a specified nucleotide in the aptamer, were used to study their interactions with thrombin. Simultaneous monitoring of both Fluorescence Anisotropy changes and electrophoretic mobility shifts upon binding of the fluorescently modified aptamer to the protein provides unique information on the specific nucleotide site of binding. T25, T20, T7 and the 3′-end were identified as the close contact sites, and T3, C15T, and the 5′-end were identified as the sites distant from the binding. This approach is highly sensitive and does not require cross-linking reactions. Studies of aptamer–protein interactions using this techniq...
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Fluorescence Anisotropy analysis for mapping aptamer protein interaction at the single nucleotide level
Journal of the American Chemical Society, 2011Co-Authors: Dapeng Zhang, Hailin WangAbstract:Structural characterization of aptamer-protein interactions is challenging and limited despite the tremendous applications of aptamers. Here we for the first time report a Fluorescence Anisotropy (FA) approach for mapping the interaction of an aptamer and its protein target at the single nucleotide level. Nine fluorescently labeled aptamers, each conjugated to a single tetramethylrhodamine at a specified nucleotide in the aptamer, were used to study their interactions with thrombin. Simultaneous monitoring of both Fluorescence Anisotropy changes and electrophoretic mobility shifts upon binding of the fluorescently modified aptamer to the protein provides unique information on the specific nucleotide site of binding. T25, T20, T7 and the 3'-end were identified as the close contact sites, and T3, C15T, and the 5'-end were identified as the sites distant from the binding. This approach is highly sensitive and does not require cross-linking reactions. Studies of aptamer-protein interactions using this technique are potentially useful for design, evolution, and modification of functional aptamers for a range of bioanalytical, diagnostic, and therapeutic applications.
Ralph Weissleder - One of the best experts on this subject based on the ideXlab platform.
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extended dynamic range imaging for noise mitigation in Fluorescence Anisotropy imaging
Journal of Biomedical Optics, 2020Co-Authors: Paolo Fumene Feruglio, Claudio Vinegoni, Ralph WeisslederAbstract:Significance: Fluorescence polarization (FP) and Fluorescence Anisotropy (FA) microscopy are powerful imaging techniques that allow to translate the common FP assay capabilities into the in vitro and in vivo cellular domain. As a result, they have found potential for mapping drug–protein or protein–protein interactions. Unfortunately, these imaging modalities are ratiometric in nature and as such they suffer from excessive noise even under regular imaging conditions, preventing accurate image-feature analysis of fluorescent molecules behaviors. Aim: We present a high dynamic range (HDR)-based FA imaging modality for improving image quality in FA microscopy. Approach: The method exploits ad hoc acquisition schemes to extend the dynamic range of individual FP channels, allowing to obtain FA images with increased signal-to-noise ratio. Results: A direct comparison between FA images obtained with our method and the standard, clearly indicates how an HDR-based FA imaging approach allows to obtain high-quality images, with the ability to correctly resolve image features at different values of FA and over a substantially higher range of Fluorescence intensities. Conclusion: The method presented is shown to outperform standard FA imaging microscopy narrowing the spread of the propagated error and yielding higher quality images. The method can be effectively and routinely used on any commercial imaging system and could be also translated to other microscopy ratiometric imaging modalities.
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Fluorescence Anisotropy imaging in drug discovery
Advanced Drug Delivery Reviews, 2018Co-Authors: Claudio Vinegoni, Paolo Fumene Feruglio, Ignacy Gryczynski, Ralph Mazitschek, Ralph WeisslederAbstract:Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence polarization/Fluorescence Anisotropy measurements are commonly employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting has proven difficult until recently. With the advent of high-resolution Fluorescence Anisotropy microscopy it is now possible to perform kinetic measurements of intracellular drug distribution and target engagement in commonly used mouse models. In this review we discuss the background, current advances and future perspectives in intravital Fluorescence Anisotropy measurements to derive pharmacokinetic and pharmacodynamic measurements in single cells and whole organs.
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rapid identification of health care associated infections with an integrated Fluorescence Anisotropy system
Science Advances, 2016Co-Authors: Ki Soo Park, Ralph Weissleder, Chen Han Huang, Kyungheon Lee, Yeong Eun Yoo, Cesar M Castro, Hakho LeeAbstract:Health care–associated infections (HAIs) and drug-resistant pathogens have become a major health care issue with millions of reported cases every year. Advanced diagnostics would allow clinicians to more quickly determine the most effective treatment, reduce the nonspecific use of broad-spectrum antimicrobials, and facilitate enrollment in new antibiotic treatments. We present a new integrated system, polarization Anisotropy diagnostics (PAD), for rapid detection of HAI pathogens. The PAD uses changes of Fluorescence Anisotropy when detection probes recognize target bacterial nucleic acids. The technology is inherently robust against environmental noise and economically scalable for parallel measurements. The assay is fast (2 hours) and performed on-site in a single-tube format. When applied to clinical samples obtained from interventional procedures, the PAD determined the overall bacterial burden, differentiated HAI bacterial species, and identified drug resistance and virulence status. The PAD system holds promise as a powerful tool for near-patient, rapid HAI testing.
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Two-Photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement
IEEE Journal of Selected Topics in Quantum Electronics, 2016Co-Authors: Claudio Vinegoni, Paolo Fumene Feruglio, John Matthew Dubach, Ralph WeisslederAbstract:Small molecule therapeutic drugs must reach their intended cellular targets (pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics). These processes are often difficult to measure in vivo due to their complexities and occurrence within single cells. It has been particularly difficult to directly measure cellular drug target binding. Fluorescence polarization is commonly used in pharmacological screening assays to measure drug-protein or protein-protein interactions. We hypothesized that Fluorescence polarization imaging could be adapted and used with fluorescently labeled drugs to measure drug target engagement in vivo . Here, we summarize recent results using two photon Fluorescence Anisotropy microscopy. Our imaging technique offers quantitative pharmacological binding information of diverse molecular interactions at the microscopic level, differentiating between bound, and unbound states. Used in combination with other recent advances in the development of novel fluorescently labeled drugs, we expect that the described imaging modality will provide a window into the distribution and efficacy of drugs in real time and in vivo at the cellular and subcellular level.
Paolo Fumene Feruglio - One of the best experts on this subject based on the ideXlab platform.
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extended dynamic range imaging for noise mitigation in Fluorescence Anisotropy imaging
Journal of Biomedical Optics, 2020Co-Authors: Paolo Fumene Feruglio, Claudio Vinegoni, Ralph WeisslederAbstract:Significance: Fluorescence polarization (FP) and Fluorescence Anisotropy (FA) microscopy are powerful imaging techniques that allow to translate the common FP assay capabilities into the in vitro and in vivo cellular domain. As a result, they have found potential for mapping drug–protein or protein–protein interactions. Unfortunately, these imaging modalities are ratiometric in nature and as such they suffer from excessive noise even under regular imaging conditions, preventing accurate image-feature analysis of fluorescent molecules behaviors. Aim: We present a high dynamic range (HDR)-based FA imaging modality for improving image quality in FA microscopy. Approach: The method exploits ad hoc acquisition schemes to extend the dynamic range of individual FP channels, allowing to obtain FA images with increased signal-to-noise ratio. Results: A direct comparison between FA images obtained with our method and the standard, clearly indicates how an HDR-based FA imaging approach allows to obtain high-quality images, with the ability to correctly resolve image features at different values of FA and over a substantially higher range of Fluorescence intensities. Conclusion: The method presented is shown to outperform standard FA imaging microscopy narrowing the spread of the propagated error and yielding higher quality images. The method can be effectively and routinely used on any commercial imaging system and could be also translated to other microscopy ratiometric imaging modalities.
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Fluorescence Anisotropy imaging in drug discovery
Advanced Drug Delivery Reviews, 2018Co-Authors: Claudio Vinegoni, Paolo Fumene Feruglio, Ignacy Gryczynski, Ralph Mazitschek, Ralph WeisslederAbstract:Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence polarization/Fluorescence Anisotropy measurements are commonly employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting has proven difficult until recently. With the advent of high-resolution Fluorescence Anisotropy microscopy it is now possible to perform kinetic measurements of intracellular drug distribution and target engagement in commonly used mouse models. In this review we discuss the background, current advances and future perspectives in intravital Fluorescence Anisotropy measurements to derive pharmacokinetic and pharmacodynamic measurements in single cells and whole organs.
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measurement of drug target engagement in live cells by two photon Fluorescence Anisotropy imaging
Nature Protocols, 2017Co-Authors: Claudio Vinegoni, Paolo Fumene Feruglio, Christian Brand, Sungon Lee, Antoinette E Nibbs, Shawn Stapleton, Sunil Shah, Ignacy GryczynskiAbstract:The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on Fluorescence Anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a Fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon Fluorescence Anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.
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Two-Photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement
IEEE Journal of Selected Topics in Quantum Electronics, 2016Co-Authors: Claudio Vinegoni, Paolo Fumene Feruglio, John Matthew Dubach, Ralph WeisslederAbstract:Small molecule therapeutic drugs must reach their intended cellular targets (pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics). These processes are often difficult to measure in vivo due to their complexities and occurrence within single cells. It has been particularly difficult to directly measure cellular drug target binding. Fluorescence polarization is commonly used in pharmacological screening assays to measure drug-protein or protein-protein interactions. We hypothesized that Fluorescence polarization imaging could be adapted and used with fluorescently labeled drugs to measure drug target engagement in vivo . Here, we summarize recent results using two photon Fluorescence Anisotropy microscopy. Our imaging technique offers quantitative pharmacological binding information of diverse molecular interactions at the microscopic level, differentiating between bound, and unbound states. Used in combination with other recent advances in the development of novel fluorescently labeled drugs, we expect that the described imaging modality will provide a window into the distribution and efficacy of drugs in real time and in vivo at the cellular and subcellular level.