Poster abstracts
Poster number 18 submitted by Golbarg M Roozbahani
Optimize delivery of DOs to live cell nuclei via active NLS
Golbarg M. Roozbahani (Postdoctoral fellow, Physics Research Building, The Ohio State University), Michael G. Poirier (Professor and Chair, Department of Physics, The Ohio State University), Carlos E. Castro (Associate Professor, Mechanical & Aerospace Eng., The Ohio State University), Wolfgang Pfeifer (Postdoctoral fellow, Mechanical & Aerospace Eng., The Ohio State University), Patrick Halley (Research Associate 1-Engineer, Mechanical & Aerospace Eng., The Ohio State University), Nathaniel L. Burge (PhD candidate, Physics Research Building, The Ohio State University)
Abstract:
DNA Origami (DO) is uniquely suited for construction of multi-functional devices for biological applications due to its unparalleled control over nanoscale geometry, ability to be functionalize with many components in a site-specific manner, biocompatibility, and triggered reconfiguration. In this work, I focused on functionalizing DNA origami structures via nuclear localization strategies to optimize their delivery to live cell nuclei. To explore this delivery, I labeled DO structures with SV40-NLS which is already fused to the human O6-alkylguanine-DNA-alkyltransfrase (hAGT, referred to as “SNAP-tag”) as a self-labeling protein tag system which takes advantage of specific activity of thioether bond between one of its Cys residues and the alkyl group of benzylguanine-modified DNA ligands (BG-DNA). I started with a particular DNA origami structure (~ 13 nm × 15 nm × 93 nm) and then tried to couple target protein-DNA conjugates to them. For this goal, a series of overhangs (OHs) were embedded at the end of each DO structures which are complementary to the BG-DNA sequence. Moreover, to quantify stability of DOs inside the nucleus, they were labeled with two spectrally distinct fluorophores at each side.
References:
1. Dang-Nguyen, T. Q.; Torres-Padilla, M. E., How Cells Build Totipotency and Pluripotency: Nuclear, Chromatin and Transcriptional Architecture. Current opinion in cell biology 2015, 34, 9-15.
2. Castro, C. E.; Kilchherr, F.; Kim, D. N.; Shiao, E. L.; Wauer, T.; Wortmann, P.; Bathe, M.; Dietz, H., A Primer to Scaffolded DNA Origami. Nature methods 2011, 8, 221-229.
3. Kalderon, D.; Roberts, B. L.; Richardson, W. D.; Smith, A. E., A Short Amino Acid Sequence Able to Specify Nuclear Location. Cell 1984, 39, 499-509.
4. Halley, PD.; Patton, RA.; Chowdhury, A.; Byrd, JC.; Castro, CE. Low-cost, simple, and scalable self-assembly of DNA origami nanostructures. Nano Research 12 (5), 1207-1215.
5. Jaekel, A.; Stegemann, P.; Saccà, B. Manipulating Enzymes Properties with DNA Nanostructures. Molecules 2019, 24, 3694.
Keywords: DNA Origami, SNAP-tag, SV40-NLS