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  1. Structural DNA nanotechnology is beginning to emerge as a widely accessible research tool to mechanistically study diverse biophysical processes. Enabled by scaffolded DNA origami in which a long single strand of DNA is weaved throughout an entire target nucleic acid assembly to ensure its proper folding, assemblies of nearly any geometric shape can now be programmed in a fully automatic manner to interface with biology on the 1–100-nm scale. Here, we review the major design and synthesis principles that have enabled the fabrication of a specific subclass of scaffolded DNA origami objects called wireframe assemblies. These objects offer unprecedented controlmore »over the nanoscale organization of biomolecules, including biomolecular copy numbers, presentation on convex or concave geometries, and internal versus external functionalization, in addition to stability in physiological buffer. To highlight the power and versatility of this synthetic structural biology approach to probing molecular and cellular biophysics, we feature its application to three leading areas of investigation: light harvesting and nanoscale energy transport, RNA structural biology, and immune receptor signaling, with an outlook toward unique mechanistic insight that may be gained in these areas in the coming decade.« less
  2. Abstract

    Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands ofmore »synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.

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