@phdthesis{Kruse2023,
  author    = {Kruse, Marlen},
  title     = {Characterization of biomolecules and their interactions using electrically controllable DNA nanolevers},
  doi       = {10.25932/publishup-57738},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-577384},
  school      = {Universit{\"a}t Potsdam},
  pages     = {100, xxii},
  year      = {2023},
  abstract  = {In this work, binding interactions between biomolecules were analyzed by a technique that is based on electrically controllable DNA nanolevers. The technique was applied to virus-receptor interactions for the first time. As receptors, primarily peptides on DNA nanostructures and antibodies were utilized. The DNA nanostructures were integrated into the measurement technique and enabled the presentation of the peptides in a controllable geometrical order. The number of peptides could be varied to be compatible to the binding sites of the viral surface proteins. Influenza A virus served as a model system, on which the general measurability was demonstrated. Variations of the receptor peptide, the surface ligand density, the measurement temperature and the virus subtypes showed the sensitivity and applicability of the technology. Additionally, the immobilization of virus particles enabled the measurement of differences in oligovalent binding of DNA-peptide nanostructures to the viral proteins in their native environment. When the coronavirus pandemic broke out in 2020, work on binding interactions of a peptide from the hACE2 receptor and the spike protein of the SARS-CoV-2 virus revealed that oligovalent binding can be quantified in the switchSENSE technology. It could also be shown that small changes in the amino acid sequence of the spike protein resulted in complete loss of binding. Interactions of the peptide and inactivated virus material as well as pseudo virus particles could be measured. Additionally, the switchSENSE technology was utilized to rank six antibodies for their binding affinity towards the nucleocapsid protein of SARS-CoV-2 for the development of a rapid antigen test device. The technique was furthermore employed to show binding of a non-enveloped virus (adenovirus) and a virus-like particle (norovirus-like particle) to antibodies. Apart from binding interactions, the use of DNA origami levers with a length of around 50 nm enabled the switching of virus material. This proved that the technology is also able to size objects with a hydrodynamic diameter larger than 14 nm. A theoretical work on diffusion and reaction-limited binding interactions revealed that the technique and the chosen parameters enable the determination of binding rate constants in the reaction-limited regime. Overall, the applicability of the switchSENSE technique to virus-receptor binding interactions could be demonstrated on multiple examples. While there are challenges that remain, the setup enables the determination of affinities between viruses and receptors in their native environment. Especially the possibilities regarding the quantification of oligo- and multivalent binding interactions could be presented.},
  language  = {en}
}
@article{KruseAltattanLauxetal.2022,
  author    = {Kruse, Marlen and Altattan, Basma and Laux, Eva-Maria and Grasse, Nico and Heinig, Lars and M{\"o}ser, Christin and Smith, David M. and H{\"o}lzel, Ralph},
  title     = {Characterization of binding interactions of SARS-CoV-2 spike protein and DNA-peptide nanostructures},
  series = {Scientific reports},
  volume    = {12},
  journal   = {Scientific reports},
  number    = {1},
  publisher = {Macmillan Publishers Limited, part of Springer Nature},
  address   = {London},
  issn      = {2045-2322},
  doi       = {10.1038/s41598-022-16914-9},
  pages     = {12},
  year      = {2022},
  abstract  = {Binding interactions of the spike proteins of the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) to a peptide fragment derived from the human angiotensin converting enzyme 2 (hACE2) receptor are investigated. The peptide is employed as capture moiety in enzyme linked immunosorbent assays (ELISA) and quantitative binding interaction measurements that are based on fluorescence proximity sensing (switchSENSE). In both techniques, the peptide is presented on an oligovalent DNA nanostructure, in order to assess the impact of mono- versus trivalent binding modes. As the analyte, the spike protein and several of its subunits are tested as well as inactivated SARS-CoV-2 and pseudo viruses. While binding of the peptide to the full-length spike protein can be observed, the subunits RBD and S1 do not exhibit binding in the employed concentrations. Variations of the amino acid sequence of the recombinant full-length spike proteins furthermore influence binding behavior. The peptide was coupled to DNA nanostructures that form a geometric complement to the trimeric structure of the spike protein binding sites. An increase in binding strength for trimeric peptide presentation compared to single peptide presentation could be generally observed in ELISA and was quantified in switchSENSE measurements. Binding to inactivated wild type viruses could be shown as well as qualitatively different binding behavior of the Alpha and Beta variants compared to the wild type virus strain in pseudo virus models.},
  language  = {en}
}