Publications by authors named "Sarit Agasti"

41 Publications

Multiplexed optical barcoding of cells photochemical programming of bioorthogonal host-guest recognition.

Chem Sci 2021 Feb 19;12(15):5484-5494. Epub 2021 Feb 19.

New Chemistry Unit, Chemistry & Physics of Materials Unit, School of Advanced Materials (SAMat), Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) Bangalore Karnataka 560064 India

Modern chemical and biological studies are undergoing a paradigm shift, where understanding the fate of individual cells, in an apparently homogeneous population, is becoming increasingly important. This has inculcated a growing demand for developing strategies that label individual cells with unique fluorescent signatures or barcodes so that their spatiotemporal trajectories can be mapped in real time. Among various approaches, light-regulated methods employing photocaged fluorophores have received particular attention, owing to their fine spatiotemporal control over labelling. However, their multiplexed use to barcode large numbers of cells for interrogating cellular libraries or complex tissues remains inherently challenging, due to the lack of multiple spectrally distinct photoactivated states in the currently available photocaged fluorophores. We report here an alternative multiplexable strategy based on optically controlled host-guest recognition in the cucurbit[7]uril (CB[7]) system that provides spatial control over the positioning of fluorophores to generate distinct barcodes in 'user-defined' cells. Using a combination of three spectrally distinct CB[7]-conjugated fluorophores and by sequentially performing cycles of photoactivation and fluorophore encoding, we demonstrate 10-color barcoding in microtubule-targeted fixed cells as well as 7-color barcoding in cell surface glycan targeted live MCF7 cells.
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http://dx.doi.org/10.1039/d0sc06860hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179588PMC
February 2021

Tricomponent Supramolecular Multiblock Copolymers with Tunable Composition via Sequential Seeded Growth.

Angew Chem Int Ed Engl 2021 Jun 10. Epub 2021 Jun 10.

New Chemistry Unit (NCU) and School of Advanced Materials (SAMat), Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore, 560064, India.

Synthesis of supramolecular block co-polymers (BCP) with small monomers and predictive sequence requires elegant molecular design and synthetic strategies. Herein we report the unparalleled synthesis of tri-component supramolecular BCPs with tunable microstructure by a kinetically controlled sequential seeded supramolecular polymerization of fluorescent π-conjugated monomers. Core-substituted naphthalene diimide (cNDI) derivatives with different core substitutions and appended with β-sheet forming peptide side chains provide perfect monomer design with spectral complementarity, pathway complexity and minimal structural mismatch to synthesize and characterize the multi-component BCPs. The distinct fluorescent nature of various cNDI monomers aids the spectroscopic probing of the seeded growth process and the microscopic visualization of resultant supramolecular BCPs using Structured Illumination Microscopy (SIM). Kinetically controlled sequential seeded supramolecular polymerization presented here is reminiscent of the multi-step synthesis of covalent BCPs via living chain polymerization. These findings provide a promising platform for constructing unique functional organic heterostructures for various optoelectronic and catalytic applications.
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http://dx.doi.org/10.1002/anie.202105342DOI Listing
June 2021

Kinetically controlled synthesis of supramolecular block copolymers with narrow dispersity and tunable block lengths.

Chem Commun (Camb) 2021 Apr 24;57(32):3937-3940. Epub 2021 Mar 24.

Supramolecular Chemistry Laboratory, New Chemistry Unit, School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore 560064, India.

Synthesis of supramolecular block copolymers (BCPs) from small monomers has been recently attempted. However, the lack of dispersity and length control of the blocky segments limits its functional outcome. Herein we demonstrate the synthesis of well-defined supramolecular BCPs with tunable block lengths by varying the monomer to seed ratio in a kinetically controlled seeded supramolecular polymerization process. Structured Illumination microscopy (SIM) and spectroscopic analyses provide structural characterization of these supramolecular BCPs, which offers various possibilities as axial organic heterostructures.
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http://dx.doi.org/10.1039/d1cc00332aDOI Listing
April 2021

Fishing for nucleic acid with a coiled hook.

Nat Chem 2021 01;13(1):5-6

New Chemistry Unit, Chemistry & Physics of Materials Unit, and School of Advanced Materials (SAMat), Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bangalore, Karnataka, India.

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http://dx.doi.org/10.1038/s41557-020-00615-9DOI Listing
January 2021

Enzyme-responsive chiral self-sorting in amyloid-inspired minimalistic peptide amphiphiles.

Nanoscale 2020 Sep 4;12(36):18692-18700. Epub 2020 Sep 4.

Chemical Biology Unit, Institute of Nano Science and Technology, Sector 64, Mohali, Punjab 160062, India.

Self-sorting is a spontaneous phenomenon that ensures the formation of complex yet ordered multicomponent systems and conceptualizes the design of artificial and orthogonally functional compartments. In the present study, we envisage chirality-mediated self-sorting in β-amyloid-inspired minimalistic peptide amphiphile (C-l/d-VFFAKK)-based nanofibers. The fidelity and stereoselectivity of chiral self-sorting was ascertained by Förster resonance energy transfer (FRET) by the judicious choice of a pyrene (Py)-hydroxy coumarin (HOCou) donor-acceptor pair tethered to the peptide sequences. Seed-promoted elongation of the homochiral peptide amphiphiles investigated by AFM image analyses and Thioflavin-T (ThT) binding study further validated the chiral recognition of the l/d peptide nanofibers. Moreover, direct visualization of the chirality-driven self-sorted nanofibers is reported using super-resolution microscopy that exhibits enantioselective enzymatic degradation for l-peptide fibers. Such enantioselective weakening of the hydrogels may be used for designing stimuli-responsive orthogonal compartments for delivery applications.
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http://dx.doi.org/10.1039/d0nr04581kDOI Listing
September 2020

Transient dormant monomer states for supramolecular polymers with low dispersity.

Nat Commun 2020 Aug 7;11(1):3967. Epub 2020 Aug 7.

Supramolecular Chemistry Laboratory, New Chemistry Unit, School of Advanced Materials (SAMat), Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore, 560064, India.

Temporally controlled cooperative and living supramolecular polymerization by the buffered release of monomers has been recently introduced as an important concept towards obtaining monodisperse and multicomponent self-assembled materials. In synthetic, dynamic supramolecular polymers, this requires efficient design strategies for the dormant, inactive states of the monomers to kinetically retard the otherwise spontaneous nucleation process. However, a generalized design principle for the dormant monomer states to expand the scope of precision supramolecular polymers has not been established yet, due to the enormous differences in the mechanism, energetic parameters of self-assembly and monomer exchange dynamics of the diverse class of supramolecular polymers. Here we report the concept of transient dormant states of monomers generated by redox reactions as a predictive general design to achieve monodisperse supramolecular polymers of electronically active, chromophoric or donor-acceptor, monomers. The concept has been demonstrated with charge-transfer supramolecular polymers with an alternating donor-acceptor sequence.
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http://dx.doi.org/10.1038/s41467-020-17799-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415150PMC
August 2020

Cooperative Supramolecular Block Copolymerization for the Synthesis of Functional Axial Organic Heterostructures.

J Am Chem Soc 2020 07 22;142(26):11528-11539. Epub 2020 Jun 22.

New Chemistry Unit and School of Advanced Materials (SAMat), Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore 560064, India.

Supramolecular block copolymerzation with optically or electronically complementary monomers provides an attractive bottom-up approach for the non-covalent synthesis of nascent axial organic heterostructures, which promises to deliver useful applications in energy conversion, optoelectronics, and catalysis. However, the synthesis of supramolecular block copolymers (BCPs) constitutes a significant challenge due to the exchange dynamics of non-covalently bound monomers and hence requires fine microstructure control. Furthermore, temporal stability of the segmented microstructure is a prerequisite to explore the applications of functional supramolecular BCPs. Herein, we report the cooperative supramolecular block copolymerization of fluorescent monomers in solution under thermodynamic control for the synthesis of axial organic heterostructures with light-harvesting properties. The fluorescent nature of the core-substituted naphthalene diimide (cNDI) monomers enables a detailed spectroscopic probing during the supramolecular block copolymerization process to unravel a nucleation-growth mechanism, similar to that of chain copolymerization for covalent block copolymers. Structured illumination microscopy (SIM) imaging of BCP chains characterizes the segmented microstructure and also allows size distribution analysis to reveal the narrow polydispersity (polydispersity index (PDI) ≈ 1.1) for the individual block segments. Spectrally resolved fluorescence microscopy on single block copolymerized organic heterostructures shows energy migration and light-harvesting across the interfaces of linearly connected segments. Molecular dynamics and metadynamics simulations provide useful mechanistic insights into the free energy of interaction between the monomers as well as into monomer exchange mechanisms and dynamics, which have a crucial impact on determining the copolymer microstructure. Our comprehensive spectroscopic, microscopic, and computational analyses provide an unambiguous structural, dynamic, and functional characterization of the supramolecular BCPs. The strategy presented here is expected to pave the way for the synthesis of multi-component organic heterostructures for various functions.
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http://dx.doi.org/10.1021/jacs.0c04404DOI Listing
July 2020

SAS-6 Association with γ-Tubulin Ring Complex Is Required for Centriole Duplication in Human Cells.

Curr Biol 2020 06 21;30(12):2395-2403.e4. Epub 2020 May 21.

School of Biology, Indian Institute of Science Education and Research, Thiruvananthapuram, Vithura, Thiruvananthapuram 695551, Kerala, India. Electronic address:

Centrioles are essential components of centrosome, the main microtubule-organizing center of animal cells required for robust spindle bipolarity [1, 2]. They are duplicated once during the cell cycle [3], and the duplication involves assembly of a cartwheel on the pre-existing centriole followed by assembly of triplet microtubules around the cartwheel [4, 5]. Although the molecular details of cartwheel formation are understood [6-13], the mechanisms initiating the formation of centriolar microtubules are not known. Here, we show that the central component of cartwheel, HsSAS-6 plays a crucial role in the formation of centriolar microtubules by interacting with the microtubule nucleation machinery, γ-tubulin ring complex (γ-TuRC) in human cells. The globular N terminus and the central coiled-coil domain of SAS-6 are required for formation of the cartwheel [7, 14], whereas the function of its C-terminal outer cartwheel region in centriole duplication remains unclear. We find that deletion of HsSAS-6 C terminus disrupts microtubule formation in daughter centriole, and as a result, cells fail to form the new centriole. Consequently, this results in mitotic cells having only two centrioles localized at a single site. Detailed molecular analyses showed that HsSAS-6 interacts with the γ-TuRC proteins and associates with the γ-TuRC at the centrosome, and furthermore, the C terminus is essential for this association. High-resolution microscopy revealed localization of the γ-TuRC protein, γ-tubulin as multiple lobes surrounding the HsSAS-6-containing central hub in the centriole. Together, the results indicate that HsSAS-6 regulates centriolar microtubule assembly by anchoring γ-TuRCs to the pro-centriole at the onset of daughter centriole formation.
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http://dx.doi.org/10.1016/j.cub.2020.04.036DOI Listing
June 2020

Synthesis and Localized Photoluminescence Blinking of Lead-Free 2D Nanostructures of Cs Bi I Cl Perovskite.

Angew Chem Int Ed Engl 2020 Jul 7;59(31):13093-13100. Epub 2020 Jul 7.

New Chemistry Unit and School of Advanced Materials, Bangalore, 560064, India.

Two-dimensional (2D) lead-free halide perovskites have generated enormous perception in the field of optoelectronics due to their fascinating optical properties. However, an in-depth understanding on their shape-controlled charge-carrier recombination dynamics is still lacking, which could be resolved by exploring the photoluminescence (PL) blinking behaviour at the single-particle level. Herein, we demonstrate, for the first time, the synthesis of nanocrystals (NCs) and 2D nanosheets (NSs) of layered mixed halide, Cs Bi I Cl , by solution-based method. We applied fluorescence microscopy and super-resolution optical imaging at single-particle level to investigate their morphology-dependent PL properties. Narrow emission line widths and passivation of non-radiative defects were evidenced for 2D layered nanostructures, whereas the activation of shallow trap states was recognized at 77 K. Interestingly, individual NCs were found to display temporal intermittency (blinking) in PL emission. On the other hand, NS showed temporal PL intensity fluctuations within localized domains of the crystal. In addition, super-resolution optical image of the NS from localization-based method showed spatial inhomogeneity of the PL intensity within perovskite crystal.
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http://dx.doi.org/10.1002/anie.202005966DOI Listing
July 2020

Self-Sorted, Random, and Block Supramolecular Copolymers via Sequence Controlled, Multicomponent Self-Assembly.

J Am Chem Soc 2020 04 13;142(16):7606-7617. Epub 2020 Apr 13.

New Chemistry Unit and School of Advanced Materials (SAMAt), Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore 560064, India.

Multicomponent supramolecular copolymerization promises to construct complex nanostructures with emergent properties. However, even with two monomeric components, various possible outcomes such as self-sorted supramolecular homopolymers, a random (statistical) supramolecular copolymer, an alternate supramolecular copolymer, or a complex supramolecular block copolymer can occur, determined by their intermolecular interactions and monomer exchange dynamics and hence structural prediction is extremely challenging. Herein, we target this challenge and demonstrate unprecedented two-component sequence controlled supramolecular copolymerization by manipulating thermodynamic and kinetic routes in the pathway complexity of self-assembly of the constitutive monomers. Extensive molecular dynamics simulations provided useful mechanistic insights into the monomer exchange rates and free energy of interactions between the monomers that dictate the self-assembly pathway and sequence. The fluorescent nature of core-substituted naphthalene diimide monomers has been further utilized to characterize the three sequences via Structured Illumination Microscopy (SIM).
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http://dx.doi.org/10.1021/jacs.0c01822DOI Listing
April 2020

Measuring mRNA translation in neuronal processes and somata by tRNA-FRET.

Nucleic Acids Res 2020 04;48(6):e32

Sagol Department of Neurobiology, University of Haifa, Haifa, Israel.

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.
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http://dx.doi.org/10.1093/nar/gkaa042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102941PMC
April 2020

Multichannel DNA Sensor Array Fingerprints Cell States and Identifies Pharmacological Effectors of Catabolic Processes.

ACS Sens 2019 12 10;4(12):3124-3132. Epub 2019 Dec 10.

UMR-S 1139, INSERM, 3PHM, Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques , Sorbonne Paris Cité, 4 avenue de l'Observatoire , 75006 Paris , France.

Cells at disease onset are often associated with subtle changes in the expression level of a single or few molecular components, making traditionally used biomarker-driven clinical diagnosis a challenging task. We demonstrate here the design of a DNA nanosensor array with multichannel output that identifies the normal or pathological state of a cell based on the alteration of its global proteomic signature. Fluorophore-encoded single-stranded DNA (ssDNA) strands were coupled via supramolecular interaction with a surface-functionalized gold nanoparticle quencher to generate this integrated sensor array. In this design, ssDNA sequences exhibit dual roles, where they provide differential affinities with the receptor gold nanoparticle as well as act as transducer elements. The unique interaction mode of the analyte molecules disrupts the noncovalent supramolecular complexation, generating simultaneous multichannel fluorescence output to enable signature-based analyte identification via a linear discriminant analysis-based machine learning algorithm. Different cell types, particularly normal and cancerous cells, were effectively distinguished using their fluorescent fingerprints. Additionally, this DNA sensor array displayed excellent sensitivity to identify cellular alterations associated with chemical modulation of catabolic processes. Importantly, pharmacological effectors, which could modulate autophagic flux, have been effectively distinguished by generating responses from their global protein signatures. Taken together, these studies demonstrate that our multichannel DNA nanosensor is well suited for rapid identification of subtle changes in a complex mixture and thus can be readily expanded for point-of-care clinical diagnosis, high-throughput drug screening, or predicting the therapeutic outcome from a limited sample volume.
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http://dx.doi.org/10.1021/acssensors.9b01009DOI Listing
December 2019

Dynamic host-guest interaction enables autonomous single molecule blinking and super-resolution imaging.

Chem Commun (Camb) 2019 Nov;55(96):14430-14433

New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bangalore, Karnataka 560064, India.

Synthetic host-guest complexes are inherently dynamic as they employ weak and reversible noncovalent interactions for their recognition processes. We strategically exploited dynamic supramolecular recognition between fluorescently labeled guest molecules to complementary cucurbit[7]uril hosts to obtain stochastic switching between fluorescence ON- and OFF-states, enabling PAINT-based nanoscopic imaging in cells and tissues.
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http://dx.doi.org/10.1039/c9cc07153aDOI Listing
November 2019

Solvent Adaptive Dynamic Metal-Organic Soft Hybrid for Imaging and Biological Delivery.

Angew Chem Int Ed Engl 2019 04 6;58(15):5008-5012. Epub 2019 Mar 6.

Chemistry and Physics of Materials Unit, School of Advanced Materials (SAMat), Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bangalore-, 560064, India.

A solvent responsive dynamic nanoscale metal-organic framework (NMOF) [Zn(1 a)(H O) ] has been devised based on the self-assembly of Zn and asymmetric bola-amphiphilic oligo-(p-phenyleneethynylene) (OPE) dicarboxylate linker 1 a having dodecyl and triethyleneglycolmonomethylether (TEG, polar) side chains. In THF solvent, NMOF showed nanovesicular morphology (NMOF-1) with surface decorated dodecyl chains. In water and methanol, NMOF exhibited inverse-nanovesicle (NMOF-2) and nanoscroll (NMOF-3) morphology, respectively, with surface projected TEG chains. The pre-formed NMOFs also unveiled reversible solvent responsive transformation of different morphologies. The flexible NMOF showed cyan emission and no cytotoxicity, allowing live cell imaging. Cisplatin (14.4 wt %) and doxorubicin (4.1 wt %) were encapsulated in NMOF-1 by non-covalent interactions and, in vitro and in vivo drug release was studied. The drug loaded NMOFs exhibited micromolar cytotoxicity.
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http://dx.doi.org/10.1002/anie.201900692DOI Listing
April 2019

Reversible encapsulations and stimuli-responsive biological delivery from a dynamically assembled cucurbit[7]uril host and nanoparticle guest scaffold.

J Mater Chem B 2018 Nov 25;6(44):7329-7334. Epub 2018 Sep 25.

New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bangalore, Karnataka 560064, India.

The positive outcome of any therapeutic molecule requires control over its delivery rate. When delivered without control, administration of large doses is required to stimulate a therapeutic effect, frequently leading to increased toxicity or undesirable side effects. Recent advances introduced "smart" materials that actively release drugs in response to environmental stimuli. Although a variety of endogenous and exogenous triggers are reported, they are either difficult to control or lack tissue penetration depth. We report here a dynamic drug delivery scaffold based on a cucurbit[7]uril (CB[7]) host and benzylammonium functionalized gold nanoparticle (AuNP) guest that utilizes a bioorthogonal small molecule to achieve therapeutic control. In addition to their ability to reach deep tissue, small molecule activation is benefitted by their external controllability. Through cell culture studies we demonstrate that the host-guest supramolecular scaffold provides a nontoxic platform that effectively encapsulates a variety of therapeutic molecules and controls the payload release upon exposure to a high-affinity competitive guest molecule. This study presents a new strategy for controlling drug release rate through the use of competitive interactions of orthogonally presented guest molecules with immediate advantages in dosage control.
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http://dx.doi.org/10.1039/c8tb01596aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7100906PMC
November 2018

Synthesis of Ultrathin Few-Layer 2D Nanoplates of Halide Perovskite CsBiI and Single-Nanoplate Super-Resolved Fluorescence Microscopy.

Inorg Chem 2018 Dec 26;57(24):15558-15565. Epub 2018 Nov 26.

The discovery of new two-dimensional (2D) perovskite halides has created sensation recently because of their structural diversity and intriguing optical properties. The toxicity of Pb-based perovskite halides led to the development of Pb-free halides. Herein, we have demonstrated a one-pot solution-based synthesis of 2D ultrathin (∼1.78 nm) few-layer (2-4 layers) nanoplates (300-600 nm lateral dimension), nanosheets (0.6-1.5 μm), and nanocrystals of layered CsBiI by varying the reaction temperature from 110 to 180 °C. We have established a mechanistic pathway for the variation of morphology of CsBiI with temperature in the presence of organic capping ligands. Further, we have synthesized the bulk powder of CsBiI by mechanochemical synthesis and liquid-assisted grinding and crystalline ingot by vacuum-sealed tube melting. 2D nanoplates and bulk CsBiI demonstrate optical absorption edge along with excitonic transition. Photoluminescence properties of individual nanoplates were studied by super-resolution fluorescence imaging, which indicated the blinking behavior down to the level of an individual CsBiI nanoplate along with its emission at the far-red region and high photostability.
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http://dx.doi.org/10.1021/acs.inorgchem.8b02887DOI Listing
December 2018

Synthetic Host-Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition.

Anal Chem 2018 10 12;90(19):11305-11314. Epub 2018 Sep 12.

New Chemistry Unit , Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) , Bangalore , Karnataka 560064 , India.

Bioorthogonal strategies are continuing to pave the way for new analytical tools in biology. Although a significant amount of progress has been made in developing covalent reaction based bioorthogonal strategies, balanced reactivity, and stability are often difficult to achieve from these systems. Alternatively, despite being kinetically beneficial, the development of noncovalent approaches that utilize fully synthetic and stable components remains challenging due to the lack of selectivity in conventional noncovalent interactions in the living cellular environment. Herein, we introduce a bioorthogonal assembly strategy based on a synthetic host-guest system featuring Cucurbit[7]uril (CB[7]) and adamantylamine (ADA). We demonstrate that highly selective and ultrastable host-guest interaction between CB[7] and ADA provides a noncovalent mechanism for assembling labeling agents, such as fluorophores and DNA, in cells and tissues for bioorthogonal imaging of molecular targets. Additionally, by combining with covalent reaction, we show that this CB[7]-ADA based noncovalent interaction enables simultaneous bioorthogonal labeling and multiplexed imaging in cells as well as tissue sections. Finally, we show that interaction between CB[7] and ADA fulfills the demands of specificity and stability that is required for assembling molecules in the complexities of a living cell. We demonstrate this by sensitive detection of metastatic cancer-associated cell surface protein marker as well as by showing the distribution and dynamics of F-actin in living cells.
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http://dx.doi.org/10.1021/acs.analchem.8b01851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6569623PMC
October 2018

Cucurbit[7]uril-Directed Assembly of Colloidal Membrane and Stimuli-Responsive Microcapsules at the liquid-liquid Interface.

Langmuir 2018 01 3;34(2):693-699. Epub 2018 Jan 3.

New Chemistry Unit and ‡Chemistry & Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) , Jakkur, Bangalore 560064, India.

Colloidal microcapsules based on supramolecular architectures feature attractive properties and offer new opportunities in diverse areas such as delivery, sensing, and catalysis. Herein, we report a new strategy to fabricate the colloidal membrane and stimuli-responsive microcapsules by utilizing cucurbit[7]uril-mediated interfacial host-guest molecular recognition. In contrast to the traditionally used cross-linking approach, this method exploits the engineered interaction between a nanoparticle ligand and cucurbit[7]uril to tune the interfacial energy and stabilize the colloidal assembly at the interface. These capsules provide a versatile platform for simultaneous encapsulation of dual cargos. Additionally, the dynamic nature of the supramolecular interactions allows triggered release of the encapsulated cargos through the orthogonal presentation of a high affinity guest molecule.
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http://dx.doi.org/10.1021/acs.langmuir.7b03554DOI Listing
January 2018

Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues.

Nano Lett 2017 10 2;17(10):6131-6139. Epub 2017 Oct 2.

Warren Alpert Medical School, Brown University , Providence, Rhode Island 02903, United States.

To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ∼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.
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http://dx.doi.org/10.1021/acs.nanolett.7b02716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658129PMC
October 2017

DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging.

Chem Sci 2017 Apr 30;8(4):3080-3091. Epub 2017 Jan 30.

Wyss Institute for Biologically Inspired Engineering , Harvard University , Boston , Massachusetts , USA . Email: ; Email:

Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled 'imager' strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in fixed cells.
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http://dx.doi.org/10.1039/c6sc05420jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380918PMC
April 2017

Quantitative super-resolution imaging with qPAINT.

Nat Methods 2016 05 28;13(5):439-42. Epub 2016 Mar 28.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.

Counting molecules in complexes is challenging, even with super-resolution microscopy. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative points accumulation in nanoscale topography (qPAINT), works independently of dye photophysics for robust counting with high precision and accuracy over a wide dynamic range. qPAINT was benchmarked on DNA nanostructures and demonstrated for cellular applications by quantifying proteins in situ and the number of single-molecule FISH probes bound to an mRNA target.
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http://dx.doi.org/10.1038/nmeth.3804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941813PMC
May 2016

Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.

Sci Transl Med 2014 Jan;6(219):219ra9

Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge Street, CPZN 5206, Boston, MA 02114, USA.

Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.
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http://dx.doi.org/10.1126/scitranslmed.3007361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063286PMC
January 2014

A photoactivatable drug-caged fluorophore conjugate allows direct quantification of intracellular drug transport.

Chem Commun (Camb) 2013 Dec;49(94):11050-11052

Center for Systems Biology, Massachusetts General Hospital/Harvard Medical School, 185 Cambridge St., Boston, MA 02114 (USA).

We report here a method that utilizes a photoactivatable drug-caged fluorophore conjugate to quantify intracellular drug trafficking processes at single cell resolution. Photoactivation is performed in labeled cellular compartments to visualize intracellular drug exchange under physiological conditions, without the need for washing, facilitating its translation into in vivo cancer models.
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http://dx.doi.org/10.1039/c3cc46089dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856557PMC
December 2013

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

J Am Chem Soc 2012 Nov 2;134(45):18499-502. Epub 2012 Nov 2.

Center for Systems Biology, Massachusetts General Hospital/Harvard Medical, Boston, 02114, United States.

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.
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http://dx.doi.org/10.1021/ja307689wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498844PMC
November 2012

Dual imaging and photoactivated nanoprobe for controlled cell tracking.

Small 2013 Jan 21;9(2):222-7. Epub 2012 Sep 21.

Center for Systems Biology, Massachusetts General Hospital/Harvard, Medical School, Boston, MA 02114, USA.

A photoactivated nanoprobe for cell labeling and tracking is demonstrated. The nanoprobe enables all targeted cells to be imaged (at 680 nm) as well as specific cells to be photoactivated using 405 nm light. Photoactivated cells can then be tracked (at 525 nm) spatiotemporally in a separate channel over prolonged periods.
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http://dx.doi.org/10.1002/smll.201201007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552149PMC
January 2013

Optochemogenetics (OCG) allows more precise control of genetic engineering in mice with CreER regulators.

Bioconjug Chem 2012 Sep 30;23(9):1945-51. Epub 2012 Aug 30.

Department of Genomic Medicine, UT MD Anderson Cancer Center, Houston, Texas 77054, USA.

New approaches that allow precise spatiotemporal control of gene expression in model organisms at the single cell level are necessary to better dissect the role of specific genes and cell populations in development, disease, and therapy. Here, we describe a new optochemogenetic switch (OCG switch) to control CreER/loxP-mediated recombination via photoactivatable ("caged") tamoxifen analogues in individual cells in cell culture, organoid culture, and in vivo in adult mice. This approach opens opportunities to more fully exploit existing CreER transgenic mouse strains to achieve more precise temporal- and location-specific regulation of genetic events and gene expression.
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http://dx.doi.org/10.1021/bc300319cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775343PMC
September 2012

Dendronized gold nanoparticles for siRNA delivery.

Small 2012 Nov 8;8(21):3253-6. Epub 2012 Aug 8.

Department of Chemistry, University of Massachusetts Amherst, 710 North Pleasant Street, Amherst, MA 01003, USA.

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http://dx.doi.org/10.1002/smll.201201141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490019PMC
November 2012

Gold nanoparticles in chemical and biological sensing.

Chem Rev 2012 May 2;112(5):2739-79. Epub 2012 Feb 2.

Department of Chemistry, University of Massachusetts, 710 North Pleasant Street, Amherst, Massachusetts 01003, USA.

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http://dx.doi.org/10.1021/cr2001178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102386PMC
May 2012

Supramolecular host-guest interaction for labeling and detection of cellular biomarkers.

Angew Chem Int Ed Engl 2012 Jan 24;51(2):450-4. Epub 2011 Nov 24.

Center for Systems Biology, Massachusetts General Hospital/Harvard Medical School, 185 Cambridge St., Boston, MA 02114, USA.

Be my guest: A supramolecular host-guest interaction is utilized for highly efficient bioorthogonal labeling of cellular targets. Antibodies labeled with a cyclodextrin host molecule bind to adamantane-labeled magnetofluorescent nanoparticles (see picture) and provide an amplifiable strategy for biomarker detection that can be adapted to different diagnostic techniques such as molecular profiling or magnetic cell sorting.
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http://dx.doi.org/10.1002/anie.201105670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309171PMC
January 2012

Controlled and sustained release of drugs from dendrimer-nanoparticle composite films.

Adv Mater 2011 Jul 15;23(25):2839-42, 2843. Epub 2011 Apr 15.

Department of Chemistry, University of Massachusetts, 710 North Pleasant St., Amherst, MA 01003, USA.

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http://dx.doi.org/10.1002/adma.201004409DOI Listing
July 2011