Single-molecule studies on DNA origami substrates
Final Report Abstract
This project at further advancing DNA origami technology toward various applications in drug discovery, biomedicine, and single-molecule biophysics. We have synthesized nanoarrays of small pharmacophores on DNA origami substrates that are displayed either as individual ligands or as fragment pairs and thereby reduced the feature size by several orders of magnitude, as compared with standard microarray techniques. Atomic force microscopy-based single-molecule detection allowed us to distinguish potent protein–ligand interactions from weak binders. Several independent binding events were directly visualized and evaluated. We applied this method to the discovery of a novel bidentate trypsin inhibitor based on benzamidine paired with the dye TAMRA, which showed tenfold enhancement of the trypsin-binding yield. This assay therefore represents a promising new tool for fragment-based drug discovery research. Building on the observation of strong non-specific protein adsorption in these experiments, we have fabricated nanoscale protein patterns over large surface areas using ordered monolayers of DNA origami nanostructures with internal cavities as molecular lithography masks. Such protein patterns have promising applications in tissue engineering and cell biology. Regular nanopatterns of four different proteins were fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin. However, this approach may enable also the large-scale patterning of other molecular species or even nanoparticles. We have also investigated DNA origami stability in low-Mg2+ buffers. DNA origami stability was found to depend on the availability of residual Mg2+ ions for the screening of electrostatic repulsion. The presence of EDTA and phosphate ions thus facilitates DNA origami denaturation by displacing Mg2+ ions from the DNA backbone and reducing the strength of the Mg2+-DNA interaction, respectively. Most remarkably, these buffer dependencies depend on DNA origami superstructure. Nevertheless, by rationally selecting buffer components and taking superstructure-dependent effects into account, the structural integrity of a given DNA origami nanostructure can be maintained in conventional buffers even at Mg2+ concentrations in the low-micromolar range. This qualifies DNA origami nanostructures for a broad spectrum of biophysical and biomedical applications incompatible with high Mg2+ concentrations. DNA origami represent powerful platforms for single-molecule investigations of protein folding studies, which, however, require strongly denaturing conditions. We have thus studied the stability of DNA origami nanostructures in the presence of the denaturing agents urea and guanidinium chloride (GdmCl) in dependence of denaturant concentration, temperature, as well as the presence of cations. At room temperature, the DNA origami were found to remain stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability was governed by variations in the melting temperature of the individual staple strands. Although GdmCl has a stronger effect on the global melting temperature, its attack resulted in less structural damage than observed for urea under conditions resulting in similar melting temperatures. This enhanced structural stability most likely originates from the ionic nature of GdmCl, which is further supported by the observed cation-induced DNA origami denaturation in moderate GdmCl concentrations. Nevertheless, their high stability at room temperature renders DNA origami nanostructures promising platforms for biophysical studies in the presence of chaotropic agents, such as single-molecule protein folding. Finally, the effects of long-term storage of preassembled staple strands on DNA origami assembly and stability was evaluated, which represents an issue of high technological relevance, especially with regard to DNA origami mass production. Our results show that staple solutions may be stored at -20°C for several years without impeding DNA origami self-assembly. Depending on DNA origami shape and superstructure, however, staple age may have negative effects on DNA origami stability under harsh treatment conditions, which we attribute to the slow accumulation of damaged nucleobases.
Publications
- Regular Nanoscale Protein Patterns via Directed Adsorption through Self- Assembled DNA Origami Masks, ACS Appl. Mater. Interfaces 8, 31239 (2016)
S. Ramakrishnan, S. Subramaniam, A.F. Stewart, G. Grundmeier, and A. Keller
(See online at https://doi.org/10.1021/acsami.6b10535) - Structural stability of DNA origami nanostructures in the presence of chaotropic agents, Nanoscale 8, 10398 (2016)
S. Ramakrishnan, G. Krainer, G. Grundmeier, M. Schlierf, and A. Keller
(See online at https://doi.org/10.1039/c6nr00835f) - Cation-induced stabilization and denaturation of DNA origami nanostructures in urea and guanidinium chloride, Small 13, 1702100 (2017)
S. Ramakrishnan, G. Krainer, G. Grundmeier, M. Schlierf, and A. Keller
(See online at https://doi.org/10.1002/smll.201702100) - On the Stability of DNA Origami Nanostructures in Low-Magnesium Buffers, Angew. Chem. Int. Ed. 57, 9470 (2018)
C. Kielar, Y. Xin, B. Shen, M.A. Kostiainen, G. Grundmeier, V. Linko, and A. Keller
(See online at https://doi.org/10.1002/anie.201802890) - Pharmacophore Nanoarrays on DNA Origami Substrates as a Single-Molecule Assay for Fragment-Based Drug Discovery, Angew. Chem. Int. Ed. 57, 14873 (2018)
C. Kielar, F.V. Reddavide, S. Tubbenhauer, M. Cui, X. Xu, G. Grundmeier, Y. Zhang, and A. Keller
(See online at https://doi.org/10.1002/anie.201806778) - Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability, Molecules 24, 2577 (2019)
C. Kielar, Y. Xin, X. Xu, S. Zhu, N. Gorin, G. Grundmeier, C. Möser, D.M. Smith, and A. Keller
(See online at https://doi.org/10.3390/molecules24142577)
