Browsing by Author "Hittelman, Walter N."

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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 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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    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.