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2 Publications

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    08/29/18 | Cell-specific chemical delivery using a selective Nitroreductase-Nitroaryl pair.
    Gruber TD, Krishnamurthy C, Grimm JB, Tadross MR, Wysocki LM, Gartner ZJ, Lavis LD
    ACS Chemical Biology. 2018 Aug 29;13(10):1888-96. doi: 10.1021/acschembio.8b00524

    The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

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    08/15/18 | Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.
    Meissner GW, Grimm JB, Johnston RM, Sutcliffe B, Ng J, Jefferis GS, Cachero S, Lavis LD, Malkesman O
    PLoS One. 2018 Aug 15;13(8):e0200759. doi: 10.1371/journal.pone.0200759

    The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

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