Synthesis of novel fluorescent probes
Many of the most widely used reporter chemistries are fluorescent or fluorescence (e.g.,"turn-on") probes. For imaging within living systems, the most useful fluoroscopes excite and emit between 600 and 800 NM. This region is biologically"quiet", with little endogenous absorption, scattering, or autofluorescence. However, there are few far-red chemical reporters. The Beatty group at OHSU is using their expertise in color chemistry to develop new fluorescent and fluorogenic probes. Much of their research in this area used the far-red fluorophore DDAO (Proc. Natl. Acad. Sci.USA 2013, ACS Chem. Biol. 2016, ACS Infect. Dis. 2016, ChemBioChem 2014) To develop novel probes, the Beatty group has synthesized far-red carbazines that can be converted into enzyme-activated fluorophores (Chem. Commun., 2016).
New technologies for mapping cellular proteins
Recent advances in imaging instrumentation and computational analysis have created new opportunities for investigating the molecular basis of diseases with remarkable detail. It is now possible to interrogate features ranging in size from angstroms to centimeters, which enables investigations into tissue architectures, neuronal connections,organelle coordination, signaling networks, and molecular organization. These are representative examples of the types of studies that would immediately benefit from a versatile technology for labeling and tracking proteins across size scales. We foresee an increased reliance on multi-color, multi-scale microscopy for investigating proteins associated with human diseases. The central obstacle that has decelerated progress in this area is the shortage of methods for labeling proteins for multi-scale microscopy.
To address this unmet need, the Beatty group at OHSU created a new concept for labeling proteins with versatile interacting peptide (VIP) tags. VIP tags combine genetically-encoded peptide tags with a palette of reporter chemistries for labeling cellular nanostructures. In 2017, Beatty and coworkers published the first demonstration of using versatile interacting peptide (VIP) tags to label cellular proteins (ChemBioChem, 2017). In that work, they described a new set of small, selective, genetically-encoded tags based on a heterodimeric coiled-coil interaction between two peptides: CoilYand CoilZ. Both CoilY or CoilZ could serve as the genetically-encoded peptidetag, which enabled them to observe two distinct cell-surface protein targets with this one heterodimeric pair. VIP tags have significant advantages over existing protein and peptide tags. They are small, target-specific, easy to use,and compatible with diverse chemical reporters, including bright organic fluorophores and quantum dots. The reporter chemistry can be selected and optimized for different applications,which makes this technology a powerful resource for dynamic and high-resolution imaging studies. In ongoing work, Prof. Beatty's team is developing a validating new VIP tags.
Fluorogenic sensors are probes that undergo a change in fluorescence emission or quantum yield in response to an enzyme reaction or other change. Sometimes fluorogenic sensors are called "turn-on" probes, because a change in their structure results in the release of a bright, fluourescent signal. The Beatty group at OHSU is using fluorogenic enzyme probes to study mycobacterial pathogenesis. They have designed probes to study sulfatase biomarkers (Proc. Natl. Acad. Sci. USA2013, ChemBioChem 2014) and esterase regulation (ChemBiochem 2014, ACSChemical Biology 2016, and ACSInfectious Disease 2016). Her group continues to develop new sensors for biochemical characterization, functional annotation, targeted imaging, and other applications.
Fluorescent reporters for High Resolution Microscopy
The advent of super resolution fluorescence microscopy (SRM) has significantly advanced biological imaging as fluorescence microscopy below the diffraction limit of light is now routinely possible. Optimization of SRM has the potential to enable the quantitation necessary to understand the spatial orientation and organization of cellular components, facilitating molecule-specific visualization at resolution great than ten times that of conventional light microscopy. Current single molecule localization SRM technology, termed photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy(STORM), relies heavily on fluorophore performance for image resolution where photoactivatable fluorescent proteins and photoswitchable small molecules are required for imaging, respectively. To date, a set of optimized fluorophores for SRM has not been designed or developed and commercially available fluorescent proteins and small molecule fluorophores are used, limiting the performance of SRM. In collaboration with the Nan lab, the Gibbs lab is developing optimized small molecule fluorophores that will enable SRM imaging in any spectral range using a combinatorial solid phase synthetic approach. We have developed a platform to quantify our novel fluorophore photoswitching properties, which aids in the selection of optimal fluorophores for SRM in all spectral ranges. We have also demonstrated the utility of STORM SRM for imaging in clinical formal in fixed paraffin embedded samples.
Bittel AM, Nickerson A, Saldivar IS, Dolman NJ, Nan X, Gibbs SL. Methodology for Quantitative Characterization of Flourophore Photoswitching to Predict Superresolution Microscopy Image Quality. Sci Rep. 2016 Jul 14;6:29687
Creech MK, Wang J, Nan, Gibbs SL. Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast
Cancer with Single Molecule Localization Microscopy. Sci. Rep. 2017 Jan 18;7:40766.
Fluorescence image-guided surgery
Surgery is one of the most common treatments prescribed for acute and chronic diseases. However, to date much of human surgery is performed without any image guidance where the surgeon has only direct visualization and palpation as their guide. Optical imaging has the potential to provide surgeons with a tool for real time image-guidance since it is the only medical imaging technology that offers non-contact, video-rate imaging. To date a number of fluorescence image-guided surgery systems are FDA approved or in clinical trials demonstrating the potential for improved surgical outcomes. However, to wield the full power of this technology, tissue- and disease-specific contrast agents are necessary that will facilitate fluorescence highlighting of important tissue to be spared and diseased tissues to be resected. The Gibbs laboratory is focused on the development of contrast agents to highlight nerve tissues to be spared as well as methods to assess tumor margin status in the operating room.
Barth CW, Schaefer JM, Rossi VM, Davis SC, Gibbs SL. Optimizing fresh specimen staining for rapid identification of tumor biomarkers during surgery. Theranostics. 2017;7(19):4722-4734. PMC5706095.
Barth CW and Gibbs SL. Direct Administration of Nerve-Specific Contrast to Improve Nerve Sparing Radical Prostatectomy.Theranostics. 2017;7(3):573-593. PMID: 28255352.
Hackman KM, Doddapaneni BS, Barth CW,Wierzbicki IH, Alani AW, Gibbs SL. Polymeric Micelles as Carriers for Nerve-Highlighting Fluorescent Probe Delivery. Mol Pharm. 2015;12(12):4386-94. PMC4674818.
Davis SC, Gibbs SL, Gunn JR, Pogue BW. Topical dual-stain difference imaging for rapid intra-operative tumor
identification in fresh specimens. Opt. Lett. 2013;38(25): 5184-7. PMC4180285.
Understanding cancer development and improved treatment strategies requires appreciation of the interaction of tens to hundreds of proteins within cells and tissues. Current protein based imaging technologies are limited in their ability to detect more than a handful of proteins on a single sample, requiring use of numerous samples to detect the multitude of vital proteins.Thus, using current techniques, it is impossible to understand complex protein interactions as they cannot be properly co-registered between different samples. To alleviate this difficulty, we are developing a cyclic immunostaining technology facilitating staining, imaging, signal removal and restaining of the same sample many times, permitting visualization of potentially hundreds of proteins in a single sample. This technology is being developed for immunofluorescence, immunohistochemistry and extension to super resolution microscopy through collaboration with Drs. Gray, Chin, Kwon, Corless, and Nan.
Fluorescently Labeled Small Molecule Therapeutics
Optimal selection of cancer therapy is challenged by current inability to effectively model and screen drugs for personalized medicine. We are developing a novel technology termed paired agent imaging in collaboration with Dr. Kenneth Tichauer at the Illinois Institute of Technology that will facilitate quantitative assessment of both drug biodistribution and therapeutic efficacy on in vitro, in vivo and explant tissue culture models. We anticipate that this technology will enable high throughput drug screening for personalized therapy selection for cancer patients.
Click Chemistry applications in imaging and proteomics
Prof.Beatty has established expertise in using bioorthogonal ligation reactions,often called "click chemistry", to image cellular proteins (MolecularBiosystems, 2011). In her prior work with Prof. David Tirrell (Caltech), she labeled cellular proteins during their biosynthesis(translation) inside cells. Proteins were labeled by incorporation of reactive unnatural amino acids, such as anazide-modified methionine analogue, and then subsequently fluorophore-modified using click chemistry to make the proteins fluorescent. Prof. Beatty described using the acopper-catalyzed azide-alkyne ligation inside E. coli (J. Am. Chem. Soc., 2005) and in mammalian cells (Angew. Chem. Int. Ed., 2006). She also demonstrated fluorophore-labeling of two distinct protein populations, which enabled changes in the proteome to be tracked over time (Bioorg. Med. Chem.Lett., 2008).
At OHSU,Prof. Beatty's group continues to use click chemistry for imaging applications and for activity-based protein profiling (ACSInfect. Dis. 2016).