Ultraschnelles Kraftmikroskop
Final Report Abstract
In order to illustrate the use of the instrument, we will explicitly describe two research high-lights, where images obtained by the AFM are the central part of the publication. In the first application the AFM was used to characterize the bottom up fabrication of nanopatterned polymers on a DNA origami structure. Bottom-up strategies to fabricate patterned polymers at the nanoscale represent an emerging field in the development of advanced nanodevices, such as biosensors, nanofluidics, and nanophotonics. DNA origami techniques provide access to distinct architectures of various sizes and shapes and present manifold opportunities for functionalization at the nanoscale with the highest precision. In this study, an in situ atom- transfer radical polymerization (ATRP) on DNA origami was conducted, yielding differently nanopatterned polymers of various heights. After cross-linking, the grafted polymeric nanostructures can even stably exist in solution without the DNA origami template. This straightforward approach allows for the fabrication of patterned polymers with low nanometer resolution, which provides access to unique DNA-based functional hybrid materials. The AFM was used for imaging the DNA origami structures alone, as well as at various stages during growth. Besides simple topographic images also elasticity imaging and adhesion imaging were performed. Moreover, the extracted cross-linked polymer was also imaged using the AFM. In the second application the AFM was used to image the compaction of nucleosomal arrays as a function of the salt concentration. Compaction of DNA in chromatin is a hallmark of the eukaryotic cell and unravelling its structure is required for an understanding of DNA involving processes. Despite strong experimental efforts, many questions concerning the DNA packing are open. In particular, it is heavily debated whether an ordered structure referred to as the “30 nm fibre” exist in vivo. Scanning probe microscopy has become a cutting edge technology for the high-resolution imaging of DNA-protein complexes. Here, we perform high-resolution atomic force microscopy of non-crosslinked chromatin arrays in liquid, under different salt conditions. A statistical analysis of the data reveals that array compaction is salt dependent in a non-monotonic fashion. A simple physical model can qualitatively explain the observed findings due to the opposing effects of salt dependent stiffening of DNA, nucleosome stability and histone-histone interactions. While for different salt concentrations different compaction states are observed, our data do not provide support for the existence of regular chromatin fibres. Our studies add new insights into chromatin structure, and with that contribute to a further understanding of the DNA condensation. Since the fibers imaged in this project were extremely fragile we used low force tapping in liquid. This particular application was one of the major reason for the choice of AFM, since peak-force tapping provides a well suited mode of operation.
Publications
- Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS, Nature Communications (2015), 6, 6161
Nagy, J. et al.
(See online at https://doi.org/10.1038/ncomms7161) - Bottom-Up Fabrication of Nanopatterned Polymers on DNA Origami by In Situ Atom-Transfer Radical Polymerization, Angewandte Chemie Internation Edition (2016), 55, 5692-5697
Tokura, Y. et al.
(See online at https://doi.org/10.1002/anie.201511761) - Atomic force microscopy of chromatin arrays reveal non-monotonic salt dependence of array compaction in solution, PLoS ONE (2017), e0173459
Krzemien, K.M., Beckers, M., Quack, S., Michaelis, J.
(See online at https://doi.org/10.1371/journal.pone.0173459) - Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes, eLIFE (2017), 6, e22510
Sundaramoorthy R. et al.
(See online at https://doi.org/10.7554/eLife.22510) - Single-molecule nucleosome remodeling by INO80 and effects of histone tails, FEBS Letters (2018), 592, 318-331
Schwarz, M. et al.
(See online at https://doi.org/10.1002/1873-3468.12973)
