Yamuna Krishnan | University of Chicago (original) (raw)

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Papers by Yamuna Krishnan

Deoxyribonucleic acid (DNA) is the molecule that carries genetic information in a cell. The field... more Deoxyribonucleic acid (DNA) is the molecule that carries genetic information in a cell. The field of DNA nanotechnology aims to use DNA as the basis for making complex nanometerscale structures. While much progress has been made in creating such nano-architectures, the use of these structures within organisms had never been attempted. Our work is focused on using a simple DNA-based pH sensor in worms to yield information about organelle activity in cells. The first of its kind, this work set up a system to use DNA as a probe in living systems.

eLife

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic interventi... more 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 Caenorhabditis elegans, with subcellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

F1000 - Post-publication peer review of the biomedical literature

Nature

A modular probe can be programmed to travel to a precise cellular destination.A modular probe can... more A modular probe can be programmed to travel to a precise cellular destination.A modular probe can be programmed to travel to a precise cellular destination.

Nature Nanotechnology

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules... more Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.DNA nanodevice can quantitatively map disulfide reduction exclusively in endosomes in C. elegans, by which the location-specific action of proteins can be addressed.

Nature Methods

It is extremely challenging to quantitate lumenal Ca2+ in acidic Ca2+ stores of the cell because ... more It is extremely challenging to quantitate lumenal Ca2+ in acidic Ca2+ stores of the cell because all Ca2+ indicators are pH sensitive, and Ca2+ transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca2+ simultaneously, CalipHluor functions as a pH-correctable Ca2+ reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca2+ changes during endosomal maturation and found a surge in lumenal Ca2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca2+ accumulation—an example of a lysosome-specific Ca2+ importer in animals. By enabling the facile quantification of compartmentalized Ca2+, CalipHluor can expand the understanding of subcellular Ca2+ importers.The DNA-based, ratiometric fluorescent reporter CalipHluor enables quantitative imaging of pH and calcium in acidic organelles with single-organelle resolution. The probe was used to identify a lysosome-specific Ca2+ importer in animals.

Proceedings of the National Academy of Sciences

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution,... more Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

Nature Chemical Biology

Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, wher... more Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome–lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.The combination of multiple fluorophores on a hybridized DNA scaffold enables the development of the reporter cHOClate, which is used to simultaneously and quantitatively image hypochlorous acid (HOCl) and pH during phagosome maturation.

RSC Biomolecular Sciences, 2012

The physicochemical properties of small molecules as well as macromolecules are modulated by solu... more The physicochemical properties of small molecules as well as macromolecules are modulated by solution pH, and DNA is no exception. Special sequences of DNA can adopt unusual conformations e.g., triplex, i-motif and A-motif, depending on solution pH. The specific range of pH for these unusual structures is dictated by the pKa of protonation of the relevant nucleobase involved in the resultant non-canonical base pairing that is required to stabilise the structure. The biological significance of these pH-dependent structures is not yet clear. However, these non-B-DNA structures have been used to design different devices to direct chemical reactions, generate mechanical force, sense pH, etc. The performance of these devices can be monitored by a photonic signal. They are autonomous and their ‘waste free’ operation cycles makes them highly processive. Applications of these devices help to increase understanding of the structural polymorphism of the motifs themselves. The design of these devices has continuously evolved to improve their performance efficiency in different contexts. In some examples, these devices have been shown to perform inside complex living systems with similar efficiencies, to report on the chemical environment there. The robust performance of these devices opens up exciting possibilities for pH-sensitive DNA devices in the study of various pH-regulated biological events.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature

Angewandte Chemie International Edition

$Research paper thumbnail of {"__content__"=>"Visualization of Ca loss from rotavirus during cell entry.", "sup"=>{"__content__"=>"2+"}}$

Journal of virology, Jan 26, 2018

Bound calcium ions stabilize many non-enveloped virions. Loss of Ca2+ from these particles appear... more Bound calcium ions stabilize many non-enveloped virions. Loss of Ca2+ from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca2+ stabilized protein (VP7). Membrane coupled, conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double layered, inner capsid particle (DLP) into the cytosol. Removal of Ca2+ in vitro strips away both outer-layer proteins; we and others have postulated that loss of Ca2+ triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca2+ sensor, the timing of Ca2+ loss from entering virions with respect to dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer-layer dissociation and DLP release. The Ca2+ sensor, placed on VP5*,...

Science

Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typic... more Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells.