Design principle for a DNA-based chemosensor that reports on MGMT activity.

<p>A short modified DNA oligomer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.t001" target="_blank">Table 1</a>) contains an <i>O</i>⁶-benzylguanine nucleoside modified with a quencher dye. Fluorescence from a neighboring fluorophore (X) is initially quenched as a result of the proximity. When MGMT repairs the alkylated base, the benzyl-quencher group is transferred to the enzyme’s active site and fluorescence emission increases.</p

Selectivity for sensing MGMT activity.

<p>When chemosensor NR-<b>1</b> was added to TK6+ cell lysates (high amounts of MGMT) an immediate increase in fluorescence was observed. Addition of probe to TK6- cell lysates (MGMT knockout) or pretreating TK6+ with 2.5 μM inhibitor PaTrin-2 (10 min, 37°C) led to no change in fluorescence with time. Final probe concentration was 50 nM and total protein used was 200 μg. Data was acquired at 37°C. Measurements were repeated 3 times. Standard deviations are provided in Figure G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.s001" target="_blank">S1 File</a>.</p

Synthesis of Dabcyl-BG nucleoside phosphoramidite 10, which was used as a monomer in preparation of chemosensors 1, 2, 3 and 4.

<p>Synthesis of Dabcyl-BG nucleoside phosphoramidite 10, which was used as a monomer in preparation of chemosensors 1, 2, 3 and 4.</p

Differential detection of MGMT activity in tumor cell lysates.

<p>Chemosensor NR-<b>1</b> was added to whole cell lysates generated from MCF-7, HT-29 and SW48 cell lines. Final spectra were acquired after a plateau in fluorescence was observed. Spectra were then subtracted from background probe fluorescence (probe alone spectrum) and normalized by total amount of protein in order to compare activity levels. Final probe concentration was 50 nM. Data were acquired at 37°C. Measurements were repeated 3 times. Standard deviations are provided in Figure G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.s001" target="_blank">S1 File</a>.</p

Sequences and optical properties of chemosensors.

<p>Sequences and optical properties of chemosensors.</p

Evaluation of MGMT inhibitors with chemosensor 1.

<p>Incubation of purified MGMT enzyme with the inhibitors BG and PaTrin-2 led to a concentration dependent decrease in observed final fluorescence intensity, indicative of MGMT inhibition. MGMT (10 nM) was incubated with inhibitor for 10 min at 37°C in 70 mM HEPES buffer (pH 7.8) containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. Final fluorescence was acquired 10 min after addition of probe (10 nM). Data were normalized to measurements without inhibitor. Each data point is the average of 3 measurements.</p

Performance of modified DNA chemosensors containing the Dabcyl-BG nucleoside.

<p>Fluorescence spectra showing the overall fold-changes in intensity observed when comparing fluorescence measured before (dashed line) and after (solid line) addition of purified MGMT protein are shown at left of each figure. Time courses (on the right) show time-dependent fluorescence increases immediately after addition of enzyme. Final probe and MGMT concentrations were 100 nM. Assays were run at 37°C in 70 mM HEPES buffer pH 7.8 containing 5 mM EDTA, 1 mM dithiothreitol and 50 μg/ml BSA. (A) chemosensor <b>1</b> containing dT^FAM, (B), chemosensor <b>2</b> containing Cy3, (C), chemosensor <b>3</b> containing dT^TMR and (D), chemosensor <b>4</b> containing perylene nucleoside. Measurements were repeated 3 times. Standard deviations are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152684#pone.0152684.t001" target="_blank">Table 1</a>.</p

Photocaged DNA-Binding Photosensitizer Enables Photocontrol of Nuclear Entry for Dual-Targeted Photodynamic Therapy

Photodynamic therapy (PDT) is a clinically approved cancer treatment that requires a photosensitizer (PS), light, and molecular oxygena combination which produces reactive oxygen species (ROS) that can induce cancer cell death. To enhance the efficacy of PDT, dual-targeted strategies have been explored where two photosensitizers are administered and localize to different subcellular organelles. To date, a single small-molecule conjugate for dual-targeted PDT with light-controlled nuclear localization has not been achieved. We designed a probe composed of a DNA-binding PS (Br-DAPI) and a photosensitizing photocage (WinterGreen). Illumination with 480 nm light removes WinterGreen from the conjugate and produces singlet oxygen mainly in the cytosol, while Br-DAPI localizes to nuclei, binds DNA, and produces ROS using one- or two-photon illumination. We observe synergistic photocytotoxicity in MCF7 breast cancer cells, and a reduction in size of three-dimensional (3D) tumor spheroids, demonstrating that nuclear/cytosolic photosensitization using a single agent can enhance PDT efficacy

Photocaged DNA-Binding Photosensitizer Enables Photocontrol of Nuclear Entry for Dual-Targeted Photodynamic Therapy

Photodynamic therapy (PDT) is a clinically approved cancer treatment that requires a photosensitizer (PS), light, and molecular oxygena combination which produces reactive oxygen species (ROS) that can induce cancer cell death. To enhance the efficacy of PDT, dual-targeted strategies have been explored where two photosensitizers are administered and localize to different subcellular organelles. To date, a single small-molecule conjugate for dual-targeted PDT with light-controlled nuclear localization has not been achieved. We designed a probe composed of a DNA-binding PS (Br-DAPI) and a photosensitizing photocage (WinterGreen). Illumination with 480 nm light removes WinterGreen from the conjugate and produces singlet oxygen mainly in the cytosol, while Br-DAPI localizes to nuclei, binds DNA, and produces ROS using one- or two-photon illumination. We observe synergistic photocytotoxicity in MCF7 breast cancer cells, and a reduction in size of three-dimensional (3D) tumor spheroids, demonstrating that nuclear/cytosolic photosensitization using a single agent can enhance PDT efficacy

Photoswitching Azo Compounds in Vivo with Red Light

The photoisomerization of azobenzenes provides a general means for the photocontrol of molecular structure and function. For applications in vivo, however, the wavelength of irradiation required for trans-to-cis isomerization of azobenzenes is critical since UV and most visible wavelengths are strongly scattered by cells and tissues. We report here that azobenzene compounds in which all four positions ortho to the azo group are substituted with bulky electron-rich substituents can be effectively isomerized with red light (630–660 nm), a wavelength range that is orders of magnitude more penetrating through tissue than other parts of the visible spectrum. When the ortho substituent is chloro, the compounds also exhibit stability to reduction by glutathione, enabling their use in intracellular environments in vivo