2 research outputs found
Virginia Orange: A Versatile, Red-Shifted Fluorescein Scaffold for Single- and Dual-Input Fluorogenic Probes
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
Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair
<p>The utility of<b> </b>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 could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-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.</p