Supported Solid Lipid Bilayers as a Platform for Single-Molecule Force Measurements

Abstract

Biocompatible surfaces are important for basic and applied research in life science with experiments ranging from the organismal to the single-molecule level. For the latter, examples include the translocation of kinesin motor proteins along microtubule cytoskeletal filaments or the study of DNA−protein interactions. Such experiments often employ single-molecule fluorescence or force microscopy.In particular for force measurements, a key requirement is to prevent nonspecific interactions of biomolecules and force probes with the surface, while providing specific attachments that can sustain loads. Common approaches to reduce nonspecific interactions include supported lipid bilayers or PEGylated surfaces. However, fluid lipid bilayers do not support loads and PEGylation may require harsh chemical surface treatments and have limited reproducibility. Here, we developed and applied a supported solid lipid bilayer (SSLB) as a platform for specific, load bearing attachments with minimal nonspecific interactions. Apart from single-molecule fluorescence measurements, anchoring molecules to lipids in the solid phase enabled us to perform force measurements of molecular motors and overstretch DNA. Furthermore, using a heating laser, we could switch the SSLB to its fluid state allowing for manipulation of anchoring points. The assay had little nonspecific interactions, was robust, reproducible, and time-efficient, and required less hazardous and toxic chemicals for preparation. In the long term, we expect that SSLBs can be widely employed for single-molecule fluorescence microscopy, force spectroscopy, and cellular assays in mechanobiology.

Single depolymerizing and transport kinesins stabilize microtubule ends

“…The motility buffer for kinesin-1 stepping assays was the same as for kinesin-8 except no KCl was added. Experiments were performed in flow cells that were either constructed using silanized, hydrophobic glass cover slides or supported solid lipid bilayers slides as described before (Bugiel et al, 2015;Sudhakar et al, 2019). The depolymerization speed and the kinesin landing rate did not significantly differ for both glass treatment.…”

Fast 3D imaging of giant unilamellar vesicles using reflected light‐sheet microscopy with single molecule sensitivity

“…Finally, the sucrose GUV solution was added and allowed to settle to the glass surface for approximately 30 min. The supported lipid bilayers were prepared using vesicle deposition 42,43 . The lipid mixture and dye ratio were the same as for the GUV preparation.…”

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter

“…without nicks and with multiple attachment points between the handles and either the magnetic bead or the glass surface (Figure 1A) (Material and Methods). The flow cell surface was passivated using a lipid bilayer strategy, which significantly reduces the non-specific adhesion of magnetic beads and proteins to the flow cell surface (58)(59)(60)(61). We provide a detailed protocol in the Materials and Methods section to establish such passivation strategy.…”