Research Projects

Nanoscale structural details of multiple endogenous proteins together inside a cell is invaluable in understanding structure-function correlation in biology. However, it is yet very challenging to obtain mostly because of various technical limitations of the commonly used ones, namely stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM). In this proposal, we aim to mitigate this by combining the emerging machine learning-based computational tools and Peptide-PAINT, a single molecule imaging technique, which achieves 5-20 nm localization precision. Moreover, using our proposed technique, we aim to investigate nanoscale distribution of the constituent proteins of transcriptional condensates, formed under both physiological and pathological conditions, namely Rhabdomyosarcoma, and to decipher the impact of these ultra-structures on pathological function. Point accumulation and imaging in nanoscale topography (PAINT) uses a transiently binding pair of either DNA oligos or peptides, which are known as docker and imager. The imager, which is covalently linked to a fluorophore, freely diffuses in the imaging solution, and transiently binds to a docker attached to a target of interest, resulting blinking in fluorescence signals at the target sites. Such approach is quite different from STORM and PALM. Also, it simplifies imaging procedure without compromising spatial resolution, and provides higher multiplexing ability. However, DNA/Peptide-PAINT is not yet commonly used for multiplex imaging because an intermediate labeling probe (ILP) is necessary to label a docker to a target of interest, and it is available only for a limited number of proteins. Therefore, we here propose to design transient binders for each target of interest which itself will act as a docker and the corresponding binder as the imager. Rhabdomyosarcoma, a common pediatric cancer, is characterized by the formation of fusion genes, PAX3FOXO1 (P3F) and PAX7FOXO1 (P7F), in which PAX3, PAX7, and FOXO1 function as essential transcription factors (TFs). The recent reports indicate that these fusion proteins have greater tendency to form condensates compared to their wild-type counterparts. Consequently, there is anomalous transcription regulation that ultimately alters the downregulation pathway, resulting in a pathogenic condition. We hypothesize that the nanoscale organization of the proteins present within transcriptional condensates, including TFs, transcription regulators, and co-activators, may significantly influence transcription regulation. This organization is likely disrupted in pathological environments, resulting in abnormal transcriptional activity. To investigate it, we propose to utilize the previously mentioned multiplex endogenous Peptide-PAINT and biochemical assays to determine any association between nanoscale distribution and transcriptional activity.