Method and Apparatus for Photon Arrival Time Interval Distribution (Paid) Analysis in Fluorescence Correlation Spectroscopy
Tech ID: 21726 / UC Case 2003-448-0
UCLA investigators have developed a new invention for monitoring molecular and macromolecular interactions in vitro and in vivo. By analyzing the photon arrival times of fluorescently-labeled molecules diffusing across a small laser excitation volume and utilizing all the available data, this new analytical method is more accurate in separating sub-populations of associated and dissociated complexes in a heterogeneous sample than existing methods.
Understanding protein interactions within a cell allows one to probe the mechanisms that control its growth, maintenance, and death. This would help lead to the development of more specific and powerful drugs to combat disease.
Several methods currently exist for studying protein-protein interactions. The yeast two-hybrid (Y2H) system is the most popular one. However, this method has several limitations, including complications associated with the use of protein fusions, incorrect protein folding, incorrect post-translational modification, and potential toxicity of the proteins of interest in yeast. Also, Y2H is based on DNA transcription, and thus, is not well suited for the study of DNA transcription factors or DNA binding proteins. Finally, Y2H is not instantaneous and cannot provide the accurate characterization that a homogenous, equilibrium binding assay can provide.
Meanwhile, ultrahigh sensitivity fluorescence detection methods have been developed that allow for the study of molecular interactions in extremely small volumes. These methods extract information regarding brightness and/or diffusion for detecting properties such as ligand-protein binding and cleavage of DNA hybrids by restriction enzymes. However, these analytical methods often fail to take advantage of all the information available in the data.
UCLA investigators have developed a new invention using photon arrival-time interval distribution (PAID) analysis for monitoring molecular and macromolecular interactions in vitro and in vivo. By analyzing the photon arrival times of fluorescently-labeled molecules excited as they diffuse across a small laser excitation volume, this invention is capable of independent and simultaneous determination of coincidence, brightness, diffusion time, and concentration of fluorophore-labeled molecules undergoing diffusion within a confocal detection volume. Unlike existing analytical methods, the PAID analysis measures brightness while retaining information on the temporal correlation of photons. This allows for a better ability to separate sub-populations of associated complexes from dissociated complexes in a heterogeneous sample.
- Drug discovery
- High throughput screening
- Mapping of signaling pathways, biochemical pathways, transcription factors interactions and all other protein-protein and protein-DNA interactions
- Simultaneously provides information on brightness, concentrations, coincidence and diffusion, leading to tighter fitting constraints and better accuracy.
- This allows for a greater ability to separate sub-populations in a heterogeneous sample and therefore to follow association/dissociation events.
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