A. Quantitative, fluorescent chemical sensors
Why develop DNA nanodevices as fluorescent reporters when a range of fluorescent proteins exist? DNA is modular, allowing the integration of independent and interdependent functionalities onto one assembly. By leveraging the 1:1 stoichiometry in DNA duplexes we can integrate multiple modules with distinct functions in precise stoichiometries onto a single DNA nanodevice. These include (1) a module that acts as fluorescent reporter of a desired analyte (2) a normalizing module for ratiometric quantitation, and (3) a targeting module that localizes the reporter in a specific organelle. We have thus made fluorescent reporters that can quantitatively image ions, reactive species as well as enzymatic activity.
1. Dan, K., Veetil, A.T., Chakraborty, K. and Krishnan, Y.* “DNA nanodevices map enzymatic activity in organelles.” Nature Nanotechnology, 2019, Accepted
2. 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. PMID: 30532082
3. 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. PMID: 26098226.
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. PMID: 23708428
5. 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. PMID: 19421220
A1. Quantitatively mapping reactive species
We expanded the technology to sense reactive species such as HOCl by mapping phagosomal HOCl, as a function of phagosome maturation in cells directly derived from human and murine blood, without further genetic modification. We showed that phagosome–lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity.
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
A2. Quantitatively mapping enzymatic cleavage in organelles
In a recent breakthrough we are very excited about, we showed that we can now selectively interrogate minor, subcellular pools of enzymes and can map the activity of these minor pools. By quantitatively mapping disulfide reduction exclusively in endosomes, we showed that disulphide exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 that are resident in late endosomes.
Dan, K., Veetil, A.T., Chakraborty, K. and Krishnan, Y.* “DNA nanodevices map enzymatic activity in organelles.” Nature Nanotechnology, 2019, Accepted
A3. Probes for long duration imaging
Our lab has developed an icosahedral DNA nanocapsule that can encapsulate quantum dots and simultaneously display ligands of defined stoichiometry and spacing on the outside. By targeting these probes to specific endocytic pathways in cells, we could track and functionally image endocytic vesicles over long durations.
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, 11, 1112-1119. PMID: 27548358.
A4. Probes for cell-specific delivery of small molecules
We also that we could release encapsulated molecules from the internal cavity of the DNA icosahedron that had been targeted to specific cells in whole organisms. Molecules can be released upon photoirradiation at single cell resolution, and even down to single endosome resolution in live organisms. We utilized this technology to study fast signaling processes such as membrane initiated steroid signaling.
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