@article {49406, title = {Cell-specific chemical delivery using a selective Nitroreductase-Nitroaryl pair.}, journal = {ACS Chemical Biology}, volume = {13}, year = {2018}, month = {2018 Aug 29}, pages = {1888-96}, abstract = {

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.

}, issn = {1554-8937}, doi = {10.1021/acschembio.8b00524}, author = {Gruber, Todd D and Krishnamurthy, Chithra and Grimm, Jonathan B and Tadross, Michael R and Wysocki, Laura M and Gartner, Zev J and Lavis, Luke D} } @article {48938, title = {Semisynthetic fluorescent pH sensors for imaging exocytosis and endocytosis.}, journal = {Nature Communications}, volume = {8}, year = {2017}, month = {2017 Nov 10}, pages = {1412}, abstract = {

The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe "semisynthetic" pH-sensitive protein conjugates with organic fluorophores, carbofluorescein, and Virginia Orange that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.

}, issn = {2041-1723}, doi = {10.1038/s41467-017-01752-5}, author = {Martineau, Magalie and Somasundaram, Agila and Grimm, Jonathan B and Gruber, Todd D and Choquet, Daniel and Taraska, Justin W and Lavis, Luke D and Perrais, David} } @article {47355, title = {Virginia Orange: A versatile, red-shifted fluorescein scaffold for single- and dual-input fluorogenic probes.}, journal = {Bioconjugate Chemistry}, volume = {27}, year = {2016}, month = {2016 Feb 17}, pages = {474-80}, abstract = {

Fluorogenic molecules are important tools for biological and biochemical research. The majority of fluorogenic compounds have a simple input-output relationship, where a single chemical input yields a fluorescent output. Development of new systems where multiple inputs converge to yield an optical signal could refine and extend fluorogenic compounds by allowing greater spatiotemporal control over the fluorescent signal. Here, we introduce a new red-shifted fluorescein derivative, Virginia Orange, as an exceptional scaffold for single- and dual-input fluorogenic molecules. Unlike fluorescein, installation of a single masking group on Virginia Orange is sufficient to fully suppress fluorescence, allowing preparation of fluorogenic enzyme substrates with rapid, single-hit kinetics. Virginia Orange can also be masked with two independent moieties; both of these masking groups must be removed to induce fluorescence. This allows facile construction of multi-input fluorogenic probes for sophisticated sensing regimes and genetic targeting of latent fluorophores to specific cellular populations.

}, issn = {1520-4812}, doi = {10.1021/acs.bioconjchem.5b00566}, author = {Grimm, Jonathan B and Gruber, Todd D and Ortiz, Gloria and Brown, Timothy A and Lavis, Luke D} }