Plasmonic hotspots for single-molecule biophysics

• Applicant: Professor Dr. Philip Tinnefeld
• Subject Area: Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics ; Biophysics ; Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 267681426

• Final Report Year: 2024

Final Report Abstract

Optical antennas, which can increase the brightness of fluorescent labels by orders of magnitude have been conventionally manufactured using lithographic methods which lack a way to specifically immobilize molecules in the zeptoliter volume of maximum fluorescence enhancement. We introduced an alternative route for the construction of these nanostructures using the self-assembly method of DNA origami which saw great success in recent years. A key area of application we envisioned was the field of single-molecule Biophysics. Here, the fluorescence enhancement could provide many advantages. For example, the antenna helps with the detection of single-molecule fluorescence in settings where the background signal of labelled species would otherwise completely overpower the molecule of interest, potentially enabling the experimenter to work at elevated, biologically relevant concentrations. Another key advantage is the increased photon output per unit time, which provides higher temporal resolution in single-molecule FRET experiments of e.g. protein dynamics. Thus, the goal of this project was to advance DNA origami nanoantennas to a state in which they could be used in exactly these settings. A first obstacle that had to be overcome was the fact that so far, the DNA origami designs that were employed did not offer enough space for the immobilization of larger molecules in the hotspot between the two plasmonic nanoparticles. This is why we completely redesigned the DNA origami structure, which resulted in improved accessibility of the plasmonic hotspot which was now able to accommodate even large biological assays and proteins, which we demonstrated by carrying out a complex bioassay in the plasmonic hotspot. In the next step, we investigated possible photophysical processes that occur at high illumination intensities, and found that certain fluorophores are more suitable for this task than others. We then used our findings and the new nanoantenna for first biophysical experiments. In collaboration with the group of Prof. Schuler (U Zürich), we immobilized an intrinsically disordered protein (IDP) labelled with a short DNA strand and a fluorescent dye in the plasmonic hotspot and observed the binding of another dye labelled IDP at high illumination intensities. After analysis using an established photon-by-photon maximum likelihood method, we were able to reproduce the previously determined lifetime of a transient encounter complex, which confirmed that biological activity is retained in the plasmonic hotspot. We then investigated the hybridization of two single-stranded DNA molecules at photon count rates exceeding 10 MHz, enabling us to determine the transition path time for this reaction to approximately 17 µs. We believe that this project significantly advanced the usability of DNA origami nanoantennas in biophysical experiments and envision many new applications for our system.

Publications

• DNA origami nanorulers and emerging reference structures. APL Materials, 8(11).
  Scheckenbach, Michael; Bauer, Julian; Zähringer, Jonas; Selbach, Florian & Tinnefeld, Philip
  
• Determining the In-Plane Orientation and Binding Mode of Single Fluorescent Dyes in DNA Origami Structures. ACS Nano, 15(3), 5109-5117.
  Hübner, Kristina; Joshi, Himanshu; Aksimentiev, Aleksei; Stefani, Fernando D.; Tinnefeld, Philip & Acuna, Guillermo P.
  
• DNA Origami Nanoantennas for Fluorescence Enhancement. Accounts of Chemical Research, 54(17), 3338-3348.
  Glembockyte, Viktorija; Grabenhorst, Lennart; Trofymchuk, Kateryna & Tinnefeld, Philip
  
• Single antibody detection in a DNA origami nanoantenna. iScience, 24(9), 103072.
  Pfeiffer, Martina; Trofymchuk, Kateryna; Ranallo, Simona; Ricci, Francesco; Steiner, Florian; Cole, Fiona; Glembockyte, Viktorija & Tinnefeld, Philip
  
• Maximizing the Accessibility in DNA Origami Nanoantenna Plasmonic Hotspots. Advanced Materials Interfaces, 9(24).
  Close, Cindy; Trofymchuk, Kateryna; Grabenhorst, Lennart; Lalkens, Birka; Glembockyte, Viktorija & Tinnefeld, Philip
  
• Salt-induced conformational switching of a flat rectangular DNA origami structure. Nanoscale, 14(21), 7898-7905.
  Hübner, Kristina; Raab, Mario; Bohlen, Johann; Bauer, Julian & Tinnefeld, Philip
  
• Gold Nanorod DNA Origami Antennas for 3 Orders of Magnitude Fluorescence Enhancement in NIR. ACS Nano, 17(2), 1327-1334.
  Trofymchuk, Kateryna; Kołątaj, Karol; Glembockyte, Viktorija; Zhu, Fangjia; Acuna, Guillermo P.; Liedl, Tim & Tinnefeld, Philip
  
• Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins. Nature Methods, 20(4), 523-535.
  Agam, Ganesh; Gebhardt, Christian; Popara, Milana; Mächtel, Rebecca; Folz, Julian; Ambrose, Benjamin; Chamachi, Neharika; Chung, Sang Yoon; Craggs, Timothy D.; de Boer, Marijn; Grohmann, Dina; Ha, Taekjip; Hartmann, Andreas; Hendrix, Jelle; Hirschfeld, Verena; Hübner, Christian G.; Hugel, Thorsten; Kammerer, Dominik; Kang, Hyun-Seo ... & Cordes, Thorben
  
• Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics. Proceedings of the National Academy of Sciences, 120(4).
  Schröder, Tim; Bohlen, Johann; Ochmann, Sarah E.; Schüler, Patrick; Krause, Stefan; Lamb, Don C. & Tinnefeld, Philip
  
• Single-Molecule FRET at 10 MHz Count Rates. Journal of the American Chemical Society, 146(5), 3539-3544.
  Grabenhorst, Lennart; Sturzenegger, Flurin; Hasler, Moa; Schuler, Benjamin & Tinnefeld, Philip