Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are ...localized in specific organs, and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell types in vivo. Here, we show that by exploiting either endogenous or synthetic receptor-ligand interactions and leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in
, with subcellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.
Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a ...custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique.
Design of selective sensors for a specific analyte in blood serum, which contains a large number of proteins, small molecules, and ions, is important in clinical diagnostics. While metal and ...polymeric nanoparticle conjugates have been used as sensors, small molecular assemblies have rarely been exploited for the selective sensing of a protein in blood serum. Herein we demonstrate how a nonspecific small molecular fluorescent dye can be empowered to form a selective protein sensor as illustrated with a thiol-sensitive near-IR squaraine (Sq) dye (λabs= 670 nm, λem= 700 nm). The dye self-assembles to form nonfluorescent nanoparticles (D h = 200 nm) which selectively respond to human serum albumin (HSA) in the presence of other thiol-containing molecules and proteins by triggering a green fluorescence. This selective response of the dye nanoparticles allowed detection and quantification of HSA in blood serum with a sensitivity limit of 3 nM. Notably, the Sq dye in solution state is nonselective and responds to any thiol-containing proteins and small molecules. The sensing mechanism involves HSA specific controlled disassembly of the Sq nanoparticles to the molecular dye by a noncovalent binding process and its subsequent reaction with the thiol moiety of the protein, triggering the green emission of a dormant fluorophore present in the dye. This study demonstrates the power of a self-assembled small molecular fluorophore for protein sensing and is a simple chemical tool for the clinical diagnosis of blood serum.
ConspectusDNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that may perform artificial functions. The pioneering developments of a DNA cube by Nadrian ...Seeman in 1991 and a DNA nanomachine by Turberfield and Yurke in 2000 spawned an entire generation of DNA nanodevices ranging from minimalist to rococo architectures. Since our first demonstration in 2009 that a DNA nanodevice can function autonomously inside a living cell, it became clear that this molecular scaffold was well-placed to probe living systems. Its water solubility, biocompatibility, and engineerability to yield molecularly identical assemblies predisposed it to probe and program biology.Since DNA is a modular scaffold, one can integrate independent or interdependent functionalities onto a single assembly. Work from our group has established a new class of organelle-targeted, DNA-based fluorescent reporters. These reporters comprise three to four oligonucleotides that each display a specific motif or module with a specific function. Given the 1:1 stoichiometry of Watson-Crick-Franklin base pairing, all modules are present in a fixed ratio in every DNA nanodevice. These modules include an ion-sensitive dye or a detection module and a normalizing dye for ratiometry that along with detection module forms a "measuring module". The third module is an organelle-targeting module that engages a cognate protein so that the whole assembly is trafficked to the lumen of a target organelle. Together, these modules allow us to measure free ion concentrations with accuracies that were previously unattainable, in subcellular locations that were previously inaccessible, and at single organelle resolution. By revealing that organelles exist in different chemical states, DNA nanodevices are providing new insights into organelle biology. Further, the ability to deliver molecules with cell-type and organelle level precision in animal models is leading to biomedical applications.This Account outlines the development of DNA nanodevices as fluorescent reporters for chemically mapping or modulating organelle function in real time in living systems. We discuss the technical challenges of measuring ions within endomembrane organelles and show how the unique properties of DNA nanodevices enable organelle targeting and chemical mapping. Starting from the pioneering finding that an autonomous DNA nanodevice could map endolysosomal pH in cells, we chart the development of strategies to target organelles beyond the endolysosomal pathway and expanding chemical maps to include all the major ions in physiology, reactive species, enzyme activity, and voltage. We present a series of vignettes highlighting the new biology unlocked with each development, from the discovery of chemical heterogeneity in lysosomes to identifying the first protein importer of Ca2+ into lysosomes. Finally, we discuss the broader applicability of targeting DNA nanodevices organelle-specifically beyond just reporting ions, namely using DNA nanodevices to modulate organelle state, and thereby cell state, with potential therapeutic applications.ConspectusDNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that may perform artificial functions. The pioneering developments of a DNA cube by Nadrian Seeman in 1991 and a DNA nanomachine by Turberfield and Yurke in 2000 spawned an entire generation of DNA nanodevices ranging from minimalist to rococo architectures. Since our first demonstration in 2009 that a DNA nanodevice can function autonomously inside a living cell, it became clear that this molecular scaffold was well-placed to probe living systems. Its water solubility, biocompatibility, and engineerability to yield molecularly identical assemblies predisposed it to probe and program biology.Since DNA is a modular scaffold, one can integrate independent or interdependent functionalities onto a single assembly. Work from our group has established a new class of organelle-targeted, DNA-based fluorescent reporters. These reporters comprise three to four oligonucleotides that each display a specific motif or module with a specific function. Given the 1:1 stoichiometry of Watson-Crick-Franklin base pairing, all modules are present in a fixed ratio in every DNA nanodevice. These modules include an ion-sensitive dye or a detection module and a normalizing dye for ratiometry that along with detection module forms a "measuring module". The third module is an organelle-targeting module that engages a cognate protein so that the whole assembly is trafficked to the lumen of a target organelle. Together, these modules allow us to measure free ion concentrations with accuracies that were previously unattainable, in subcellular locations that were previously inaccessible, and at single organelle resolution. By revealing that organelles exist in different chemical states, DNA nanodevices are providing new insights into organelle biology. Further, the ability to deliver molecules with cell-type and organelle level precision in animal models is leading to biomedical applications.This Account outlines the development of DNA nanodevices as fluorescent reporters for chemically mapping or modulating organelle function in real time in living systems. We discuss the technical challenges of measuring ions within endomembrane organelles and show how the unique properties of DNA nanodevices enable organelle targeting and chemical mapping. Starting from the pioneering finding that an autonomous DNA nanodevice could map endolysosomal pH in cells, we chart the development of strategies to target organelles beyond the endolysosomal pathway and expanding chemical maps to include all the major ions in physiology, reactive species, enzyme activity, and voltage. We present a series of vignettes highlighting the new biology unlocked with each development, from the discovery of chemical heterogeneity in lysosomes to identifying the first protein importer of Ca2+ into lysosomes. Finally, we discuss the broader applicability of targeting DNA nanodevices organelle-specifically beyond just reporting ions, namely using DNA nanodevices to modulate organelle state, and thereby cell state, with potential therapeutic applications.
Deep tissue bioimaging with three-photon (3P) excitation using near-infrared (NIR) light in the second IR window (1.0-1.4 μm) could provide high resolution images with an improved signal-to-noise ...ratio. Herein, we report a photostable and nontoxic 3P excitable donor-π-acceptor system (GMP) having 3P cross-section (σ3 ) of 1.78×10(-80) cm(6) s(2) photon(-2) and action cross-section (σ3 η3 ) of 2.31×10(-81) cm(6) s(2) photon(-2) , which provides ratiometric fluorescence response with divalent zinc ions in aqueous conditions. The probe signals the Zn(2+) binding at 530 and 600 nm, respectively, upon 1150 nm excitation with enhanced σ3 of 1.85×10(-80) cm(6) s(2) photon(-2) and σ3 η3 of 3.33×10(-81) cm(6) s(2) photon(-2) . The application of this probe is demonstrated for ratiometric 3P imaging of Zn(2+) in vitro using HuH-7 cell lines. Furthermore, the Zn(2+) concentration in rat hippocampal slices was imaged at 1150 nm excitation after incubation with GMP, illustrating its potential as a 3P ratiometric probe for deep tissue Zn(2+) ion imaging.
Neutralization of pathogens by phagocytic immune cells requires the biogenesis of a compartmentalized hotspot of reactive species called the phagosome. One of these reactive species is hypochlorous ...acid (HOCl), produced by the enzyme myeloperoxidase (MPO) after the phagosome fuses with the lysosome. Mapping HOCl during phagosome maturation can report on pathogen killing and offer insights into regulation of MPO activity, mechanisms of resistance and host-pathogen interactions. However, this has been difficult because of a lack of a suitable method to chemically map a transient organelle with pH fluctuations like the phagosome. Here, we detail a protocol for quantifying HOCl dynamics in phagosomes using a fluorescent DNA-based reporter. Compared to traditional methods of visualizing HOCl or measuring MPO activity, this method offers sub-cellular spatial resolution and the capacity to assay HOCl production with single cell resolution.
Antigen-binding fragments of antibodies are biotechnologically useful agents for decorating drug delivery systems, for blocking cell-surface receptors in cell culture, for recognizing analytes in ...biosensors, and potentially as therapeutics. They are typically produced by enzymatic digestion of full antibodies and isolated from the undesirable fragment crystallizable (Fc) by affinity chromatography using Protein-A columns. However, while Protein-A has a strong “classical” interaction with Fc fragments, it can also more weakly bind to an “alternative” site on the heavy chain variable region of antigen-binding fragments. As such, purifying small amounts of antibody fragments by Protein-A chromatography can result in low yield. Moreover, loading larger amounts of antibody fragments onto a Protein-A column can result in poor separation, because of competition of Fc and antigen-binding fragments for immobilized Protein-A. This study demonstrates that Protein-A-based homogeneous scavenging resolves this issue by precisely controlling the stoichiometry of Protein-A to Fc fragments, something that is not possible for conventional flow-type systems, such as affinity chromatography.
The development of molecular probes for the detection and imaging of biological thiols is a major step forward diagnosing various types of diseases. Previously reported thiol imaging strategies were ...mainly based on a single mode of imaging with a limited application
. In this work, we introduced an unsymmetrical near-infrared (NIR) squaraine dye (USq) as an exogenous contrast agent for photoacoustic and fluorescence bimodal imaging of thiol variations in live animals. USq exhibits a narrow absorption band at 680 nm that generates a photoacoustic signal and a strong NIR emission at 700 nm (
= 0.27), which is applicable for deep tissue optical imaging. Both photoacoustic and fluorescence signals could selectively disappear in the presence of different thiols. Through
and
imaging studies, unique imaging capability of USq was demonstrated, and the effect of food uptake on the increased level of aminothiols in blood was confirmed.
Passive endocytosis in model protocells Zhang, Stephanie J; Lowe, Lauren A; Anees, Palapuravan ...
Proceedings of the National Academy of Sciences - PNAS,
06/2023, Letnik:
120, Številka:
24
Journal Article
Recenzirano
Odprti dostop
Semipermeable membranes are a key feature of all living organisms. While specialized membrane transporters in cells can import otherwise impermeable nutrients, the earliest cells would have lacked a ...mechanism to import nutrients rapidly under nutrient-rich circumstances. Using both experiments and simulations, we find that a process akin to passive endocytosis can be recreated in model primitive cells. Molecules that are too impermeable to be absorbed can be taken up in a matter of seconds in an endocytic vesicle. The internalized cargo can then be slowly released over hours, into the main lumen or putative cytoplasm. This work demonstrates a way by which primitive life could have broken the symmetry of passive permeation prior to the evolution of protein transporters.
Lysosomal calcium (Ca
) release is critical to cell signaling and is mediated by well-known lysosomal Ca
channels. Yet, how lysosomes refill their Ca
remains hitherto undescribed. Here, from an RNA ...interference screen in
, we identify an evolutionarily conserved gene,
, that facilitates lysosomal Ca
entry in
and mammalian cells. We found that its human homolog TMEM165, previously designated as a Ca
/H
exchanger, imports Ca
pH dependently into lysosomes. Using two-ion mapping and electrophysiology, we show that TMEM165, hereafter referred to as human LCI, acts as a proton-activated, lysosomal Ca
importer. Defects in lysosomal Ca
channels cause several neurodegenerative diseases, and knowledge of lysosomal Ca
importers may provide previously unidentified avenues to explore the physiology of Ca
channels.
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