Browsing by Author "Zimak, Jan"

Now showing 1 - 5 of 5
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    Configuring robust DNA strand displacement reactions forﾠin situﾠmolecular analyses
    (Oxford University Press, 2012) Duose, Dzifa Y.; Schweller, Ryan M.; Zimak, Jan; Rogers, Arthur R.; Hittelman, Walter N.; Diehl, Michael R.
    The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterativein situﾠlabeling and erasing of markers on cells.
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    Multiplexed in situ molecular analyses and programmable molecular probes for regulated single amplification
    (2018-12-11) Diehl, Michael; Zimak, Jan; Schweller, Ryan; Samson, Edward B.; Duose, Dzifa Y.; Rice University; United States Patent and Trademark Office
    The present invention generally relates to methods for detecting a target in a sample; methods for modulating the reporting intensity of a labeled target in a sample of fixed cells or tissues; methods for detecting the location of at least two targets in a sample; and related compositions.
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    Multiplexed In Situ Immunofluorescence Using Dynamic DNA Complexes
    (Wiley, 2012) Schweller, Ryan M.; Zimak, Jan; Duose, Dzifa Y.; Qutub, Amina A.; Hittelman, Walter N.; Diehl, Michael R.
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    Multiplexed Spatial Analyses in situ and in living cells
    (2015-12-04) Zimak, Jan; Diehl, Michael R; Qutub, Amina A; Wagner, Daniel S
    High-content spatial analyses are critical to understanding the structural organization and dynamics of many complex biological processes. Increasing the number of cellular components that can be visualized will help delineate the functions of many interacting and competing cellular pathways. However, the physical limitations of spectral bandwidth and the experimental difficulty of genomic manipulation have hampered traditional approaches to multiplex molecular analyses in both fixed samples and live cells. The programmable and predictable nature of the DNA molecule makes it a tantalizing candidate for an engineering tool to help alleviate some of these limitations. This thesis seeks to harness both the chemical and biological utility of DNA as a building block to multiplex the color and control the number and location of fluorescent reporters in biological samples. First in the context of in situ immunofluorescence imaging of fixed cells or tissues, And second in the context of live-cell imaging of genomically engineered cells. In the first case, by utilizing the stand displacement chemical reaction between dynamic DNA complexes and DNA-conjugated antibodies we selectively couple fluorophores to, and then remove them, from their protein targets. We leverage this mechanism to facilitate multiple sequential round of fluorescence microscopy where the same color dye molecules are used reiteratively to visualize different antibody-tagged markers. By optimizing the DNA-antibody conjugation chemistry and incubation protocol we now routinely perform 9 marker analyses of paraffin-fixed tissue sections with these DNA probes. Then automating the sequence design process enabled more complex probe designs to be use for balancing marker levels appropriately for hyperspectral imaging experiments. Here, discrete and reconfigurable control over amplification gains, greatly improved the spectral un-mixing of different antibody signals. Secondly, we focus on dissecting network-level functions of cytoskeletal regulatory proteins during epithelial cell polarization and morphogenesis. DNA-based STORM microscopy revealed that a scaffold protein, IQGAP1, associates with specialized actin filaments within cell-cell junctions and with basket-like structures in the basal actin cortex of normal epithelial cells. This work uses IQGAP1 as a platform, as it lies at the nexus of cell signaling and cytoskeletal regulatory networks. We construct multi-gene systems that simultaneously sense and control intracellular expression levels of IQGAP1 and track the actin cytoskeleton. By combining novel molecular biology techniques to manipulate the DNA in live cells. We use a barcoded self-assembly technique to construct large vectors that contain several transcriptional elements. These multi-gene systems are then stably incorporated into cells engineered with genomic ‘landing pads’ using locus-specific integration. Finally, we demonstrate functional circuits by linearly controlling intracellular IQGAP1 levels. These results will support future single-cell and multiplexed population-level analyses of IQGAP1 functions in epithelial cells, allow us to study IQGAP1 recruitment to epithelial cell-cell junctions and to examine how it influences cellular transitions.
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    Programming in situ immunofluorescence intensities through interchangeable reactions of dynamic DNA complexes
    (Wiley-VCH Verlag, 2012) Zimak, Jan; Schweller, Ryan M.; Duose, Dzifa Y.; Hittelman, Walter N.; Diehl, Michael R.
    The regulation of antibody reporting intensities is critical to various in situ fluorescence imaging analyses. While such control is often necessary to visualize sparse molecular targets, the ability to tune marker intensities is also essential for highly multiplexed imaging strategies where marker reporting levels must be tuned to both optimize dynamic detection ranges and minimize crosstalk between different signals. Existing chemical amplification approaches generally lack such control. Here, we demonstrate that linear and branched DNA complexes can be designed to function as interchangeable building blocks that can be assembled into organized, fluorescence reporting complexes. We show that the ability to program DNA strand displacement reactions between these complexes offer new opportunities to deterministically tune the number of dyes that are coupled to individual antibodies in order to both increase and controllably balance marker levels within fixed cells.