Research Interests:
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Nucleic acid-based Molecular Devices
Our lab elicits new functions from DNA beyond that of its traditional role as Nature’s genetic material. DNA nanodevices are nanoscale assemblies, formed from a collection of synthetic DNA strands, that can have artificial function engineered into them. We have created quantitative imaging technology that uses DNA nanodevices as fluorescent reporters to map second messengers in real time in cells and in vivo. Until our innovation, it was not at all obvious whether such DNA nanodevices could function inside a living cell without being interfered with, or interfering with, the cells own networks of DNA control.
We have developed a range of strategies around this concept to interrogate diverse biological processes not previously amenable to analysis. We can now transform virtually any detection chemistry for various analytes into quantitative detection chemistries and localize that detection tissue-specifically in specific pre-designated subcellular locations in live organisms. Given the powerful ability to engineer a range of functionalities into DNA nanodevices, our lab seeks to create powerful biological imaging tools where none exist and unplug decades-old bottlenecks that have prevented quantitative measurements required to answer questions in systems biology.
Quantitative Functional Imaging
The versatile, functional imaging technology developed by us uses self-assembled DNA nanostructures to quantitatively image second messengers in real time, in living cells (1) and genetic model organisms (3). Why develop DNA nanodevices as fluorescent reporters when a range of fluorescent proteins exist? DNA is a modular scaffold, allowing the integration of independent and interdependent functionalities onto one assembly. By harnessing the modularity of DNA, we continuously develop two halves of a universal in vivo chemical imaging platform. One halfdeals with creating "measuring modules" for specific analytes. It uses a DNA scaffold to display both (i) analyte detection chemistries and (ii) normalizing dyes to measure analyte concentration (1-3). The other half creates "targeting modules" for specific organelles. Here, organelle trafficking motifs are incorporated onto DNA nanodevices to enable their localization in a designated area of the cell (1). Together the two halves now allow one to measure analyte concentrations with accuracies that were previously unattainable in subcellular locations that were previously inaccessible.
1. Modi, S.; Swetha, M.G.; Goswami, D.; Gupta, G.D.; Mayor, S.; Krishnan, Y.* "A DNA nanomachine that maps spatial and temporal pH changes in living cells." Nature Nanotechnology, 2009, 4, 325-330. PMID: 19421220.
2. Saha, S., Prakash, V., Halder, S., Chakraborty, K., Krishnan, Y.* “A pH-insensitive DNA nanodevice quantifies chloride in organelles of living cells.” Nature Nanotechnology, 2015, 10, 645 – 651. PMID: 26098226.
3. Chakraborty, K., Leung, K., Krishnan, Y.* "High lumenal chloride in the lysosome is critical for lysosome function." eLife, 2017, 6, e28862. PMID: 28742019.
Expanding the technology in living systems
We recently expanded the capability of this technology in several ways. We showed that we could use two spectrally different pH reporters to map pH in two different organelles within the same cell at the same time (4). We also showed that we could simultaneously measure two analytes in the same organelle, retaining information on concentrations of both analytes with single organelle addressability (5,6). We expanded the capability of the technology to sense not just ions, but reactive species such as HOCl (7). We also showed that we could image organelles other those along the endolysosomal pathway, e.g., the trans Golgi network, and the phagosome (4, 5).
4. Modi, S.; Nizak, C.; Surana, S.; Halder, S.; Krishnan, Y.* “Two DNA nanomachines map pH of intersecting endocytic pathways.” Nature Nanotechnology, 2013, 8, 459-467. PMID: 23708428.
5. Leung, K., Chakraborty, K., Saminathan, A., Krishnan, Y.* “A DNA Nanomachine chemically resolves lysosomes in live cells.” Nature Nanotechnology, 2018,10.1038/s41565-018-0318-5. PMID: 30510277.
6. Narayanaswamy, N., Chakraborty, K., Saminathan, A., Zeichner, E., Leung, K., Devany, J., Krishnan, Y.* “A pH-correctable DNA-based fluorescent reporter for organellar calcium.” Nature Methods, 2019, 16, 95-102. PMID: 30532082
7. Thekkan, S., Jani, M. S., Cui, C., Zhou, G., Becker, L.*, Krishnan, Y.* "A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome." Nature Chemical Biology, 2018, DOI: 10.1038/s41589-018-0176-3. PMID: 30531966
Cargo Delivery and Long Duration Live Imaging
We have developed an icosahedral DNA nanocapsule that can harbor molecular cargo as a payload on the inside, while displaying ligands of defined stoichiometry and spacing on the outside (8,9). This allows one target these nanodevices to specific endocytic pathways in cells, allowing us to track and functionally image endocytic vesicles over long durations (10).
8. Bhatia, D., Arumugam, S., Nasilowski, M., Joshi, H., Wunder, C., Chambon, V., Prakash, V., Grazon, C., Nadal, B., Maiti, P.K., Johannes, L.*, Dubertret, B.*, Krishnan, Y.* "Quantum dot-loaded monofunctionalized DNA Icosahedra for single particle tracking of endocytic pathways." Nature Nanotechnology, 2016, in press.
9. Veetil, A.T., Chakraborty, K., Xiao, K., Minter, M. R., Sisodia, S.S., Krishnan, Y.* "Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules." Nature Nanotechnology, 2017, 12, 1183-89. PMID: 28825714.
10. Veetil, A. T, Jani, M. S, Krishnan, Y.* "Chemical control over membrane-initiated steroid signaling using DNA nanocapsules." Proc. Natl. Acad. Sci. U.S.A., 2018, 115, 9432-9437. PMID: 29531078.
Future Directions
The next wave of DNA nanotechnology is seeing a gamut of DNA and RNA devices deployed to probe, program and re-program living cells, or even whole organisms. Many of these use the molecular delivery framework established by our lab. Because DNA is highly immunogenic, the extraneous introduction of DNA into higher organisms could lead to very complicated scenarios, very fast. Peruse our reviews that outline (i) a roadmap articulating how DNA nanodevices can leverage the immune system of higher organisms (ii) the emergence of DNA/RNA based imaging technologies in biology and (iii) the big challenges ahead for nanostructured DNA/RNA technologies.
11. Surana, S.; Shenoy, A.R.; Krishnan, Y.* “Designing DNA nanodevices for compatibility with the immune system of higher organisms.” Nature Nanotechnology, 2015, 10, 741-747. PMID: 21654639.
12. Chakraborty, K.; Veetil, A.T.; Jaffrey, S. R.*; Krishnan, Y.* “Nucleic acid-based nanodevices in biological imaging.” Annual Reviews of Biochemistry, 2015, 85, 349-73. PMID: 27294440.