Biosensors Environmental Dyes Quantum Dots Remote Detection
The core of the TCNP program is the development of biosensors that display reversible changes in their optical properties in response to 5 major classes of biological process:
So far we have developed the first examples of these tools as single chain antibodies (Fluorescent Activating Peptides-FAPs) that bind to and activate fluorescence of nonfluorescent molecules such as malachite green and thiazole orange. These single-chain antibodies can be expressed as fusion constructs with cellular proteins, and then the proteins are illuminated by addition of the complementary dye molecule. Our first tools have been used to illuminate proteins on the surface of the cell (see Figure 1A). Use of cell-impermeant dyes can dramatically increase the discrimination of the cell-surface molecules compared to typical methods like green-fluorescent protein fusion tagging. We have shown that routine analysis of these modules with a spectral confocal system enables us to detail the optical and biophysical behavior of the biosensor modules (see Figure 1B).
By selecting from a yeast surface display library containing the naive human antibody repertoire, we are able to select clones that specifically bind to nonfluorescent dyes. Sorting of these yeast based on fluorescence activation in the dye channel allows selection and evolution of brighter and higher affinity clones with improved capability to activate the fluorogen to a fluorescent state. These FAP modules can be genetically expressed in yeast secretion systems for solution characterization of affinity and spectral properties. One shortcoming of the solution characterization, however, is that it reveals no information about site-selectivity or access of the fluorogenic dyes to the modules at different microenvironments in the cell, or about the specificity of the fluorogen for the expressed module. The investigation of the properties of these materials on living cells, with advanced multi-laser microscopy reveals this in a way that biochemical investigations would not.
Because of the exquisite specificity afforded by an antibody interaction with these small molecules, allosteric interactions can influence the binding site of the FAP. Ideally, these changes would result in changes of the electrosteric interactions with the dye molecule. These changes modulate the spectral and other optical properties of the bound activated dye, as well as the affinity of the dye-FAP interactions. In order to develop kinase sensors, for example, we are placing kinase substrate sites at a number of distinct locations in one of our developed FAP modules with hopes of discovering sites that convey phosphorylation dependent properties to the bound fluorogen. We are performing solution characterization of these modules under biochemical conditions that cause phosphorylation, which involves creating and isolating secreted forms of each of these molecules.
