Structural Transformation of Wireframe DNA Origami via DNA Polymerase Assisted Gap-Filling

Abstract

The programmability of DNA enables constructing nanostructures with almost any arbitrary shape, which can be decorated with many functional materials. Moreover, dynamic structures can be realized such as molecular motors and walkers. In this work, we have explored the possibility to synthesize the complementary sequences to single-stranded gap regions in the DNA origami scaffold cost effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions. This reduced the number of staple strands needed to determine the shape and size of the final structure after gap filling. For this, several DNA polymerases and single-stranded binding (SSB) proteins were tested, with T4 DNA polymerase being the best fit. The structures could be folded in as little as 6 min, and the subsequent optimized gap-filling reaction was completed in less than 3 min. The introduction of flexible gap regions results in fully collapsed or partially bent structures due to entropic spring effects. Finally, we demonstrated structural transformations of such deformed wireframe DNA origami structures with DNA polymerases including the expansion of collapsed structures and the straightening of curved tubes. We anticipate that this approach will become a powerful tool to build DNA wireframe structures more material-efficiently, and to quickly prototype and test new wireframe designs that can be expanded, rigidified, or mechanically switched. Mechanical force generation and structural transitions will enable applications in structural DNA nanotechnology, plasmonics, or single-molecule biophysics.

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

“…Recombinases, topoisomerases, helicases, ligases, and relaxases have been utilized to induce structural and mechanical changes in nucleic acid devices ( 20–26 ). DNA polymerases have been used to induce structural changes in DNA nanostructures ( 20 , 27 ), replicate nanostructures ( 28 ), and build a range of dynamic chemical reaction networks ( 29–35 ). RNA polymerases, such as T7 RNA polymerase, have been used to transcribe self-assembling RNA nanostructures ( 36–39 ) and materials ( 28 ), drive molecular motion and structural changes in DNA nanostructures ( 40 , 41 ), and engineer dynamic chemical reaction networks ( 42–44 ).…”

“…RNA polymerases, such as T7 RNA polymerase, have been used to transcribe self-assembling RNA nanostructures ( 36–39 ) and materials ( 28 ), drive molecular motion and structural changes in DNA nanostructures ( 40 , 41 ), and engineer dynamic chemical reaction networks ( 42–44 ). These applications take advantage of specific reactions that occur between nucleic acids and proteins, but often unintended interactions arise when coupling DNA devices and DNA binding proteins which can hamper device functionality ( 20 , 27 , 40 ). To continue to expand the functionality of nucleic acid devices, an understanding of the non-specific or unintended interactions between DNA nanostructures and DNA binding proteins is imperative.…”

Increasing Complexity in Wireframe DNA Nanostructures

“…For example, Matthies et al [67] demonstrated a DNA truss with triangulated mesh. Later, Agarwal et al [68] from the same research group showed that DNA polymerase can be employed to fill the single-stranded gaps in a truss structure with dsDNA after the assembly owing to the better accessibility compared to lattice-based DNA origami with closely packed helices. The triangulated meshes in both of these works were realized using a custom-developed script called "k-route" [67].…”

Dynamic DNA Origami Devices: from Strand-Displacement Reactions to External-Stimuli Responsive Systems

“…By applying toehold-mediated strand-displacement processes, multiple rounds of conformational switching could be demonstrated. In addition to using strand displacement, the needed DNA trigger can also be grown using DNA polymerase, as described by Agarwal et al [ 72 ]. They demonstrated the straightening and rigidifying of a deformed wireframe DNA origami having ssDNA gaps by growing a complementary gap-filling strand on site by DNA polymerase.…”