Probing tethered targets of a single biomolecular complex with atomic force microscopy

Abstract

DNA origami shows tremendous promise as templates for the assembly of nano-components and detection of molecular recognition events. So far, the method of choice for evaluating these structures has been atomic force microscopy (AFM), a powerful tool for imaging nanoscale objects. In most cases, tethered targets on DNA origami have proven to be highly effective samples for investigation. Still, while maximal assembly of the nanostructures might benefit from the greatest flexibility in the tether, AFM imaging requires a sufficient stability of the adsorbed components. The balance between the tether flexibility and sample stability is a major, poorly understood, concern in such studies. Here, we investigated the dependence of the tethering length on molecular capture events monitored by AFM. In our experiments, single biotin molecules were attached to DNA origami templates with various linker lengths of thymidine nucleotides, and their interaction with streptavidin was observed with AFM. Our results show that the streptavidin-biotin complexes are easily detected with short tethered lengths, and that their morphological features clearly change with the tethering length. We identify the functionally useful tether lengths for these investigations, which are also expected to prove useful in the construction and further application of DNA origami in bio-nanotechnology studies.

From Nonfinite to Finite 1D Arrays of Origami Tiles

“…Because we frequently observe “failure products”, it is useful to determine whether the incorporated block is the correct type by marking one of the two structures. Streptavidin has a long history of use as a marker in DNA nanotechnology because the streptavidin–biotin complex has one of the highest noncovalent binding strengths in nature, with an associated free energy of 18 kcal/mol. − In order to make it easier to identify the blocks, one or two arms of the CLS can be modified with two closely spaced biotinylated staples during individual CLS assembly, then labeled through binding with streptavidin as a high contrast marker before imaging with AFM. − For example, a single streptavidin marker on one arm enables us to identify the CLS1 components of the array and their orientation along all three axes. To break the symmetry of the CLS, two biotinylated staples were positioned between the seventh and the eighth helix of the 12 helixes in the right arm counting from the “up arm” as shown in Figure l.…”

Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed

“…These virtual issues are labeled on the JMR website (https://onlinelibrary.wiley.com/page/journal/10991352/homepage/VirtualIssuesPage.html), which provides quick links to published articles . This new special virtual issue contained eight research articles Fifth AFMBioMed Conference 2013 Shanghai, China, 8‐11 MayConference chair: Dr. Jun Hu (Shanghai Inst.…”