Reconfigurable pH-Responsive DNA Origami Lattices

DNA nanotechnology enables straightforward fabrication of user-defined and nanometer-precise templates for a cornucopia of different uses. To date, most of these DNA assemblies have been static, but dynamic structures are increasingly coming into view. The programmability of DNA not only allows for encoding of the DNA object shape but also it may be equally used in defining the mechanism of action and the type of stimuli-responsiveness of the dynamic structures. However, these “robotic” features of DNA nanostructures are usually demonstrated for only small, discrete, and device-like objects rather than for collectively behaving higher-order systems. Here, we show how a large-scale, two-dimensional (2D) and pH-responsive DNA origami-based lattice can be assembled into two different configurations (“open” and “closed” states) on a mica substrate and further switched from one to the other distinct state upon a pH change of the surrounding solution. The control over these two configurations is achieved by equipping the arms of the lattice-forming DNA origami units with “pH-latches” that form Hoogsteen-type triplexes at low pH. In short, we demonstrate how the electrostatic control over the adhesion and mobility of the DNA origami units on the surface can be used both in the large lattice formation (with the help of directed polymerization) and in the conformational switching of the whole lattice. To further emphasize the feasibility of the method, we also demonstrate the formation of pH-responsive 2D gold nanoparticle lattices. We believe this work can bridge the nanometer-precise DNA origami templates and higher-order large-scale systems with the stimuli-induced dynamicity.


Materials
All chemicals were purchased from commercial suppliers and used as received unless otherwise noted. For preparation of the DNA origami unit, the circular single-stranded p7249 scaffold (c = 100 nM) was purchased from Tilibit Nanosystems and the single-stranded staple strands from Integrated DNA Technologies. 50× TAE buffer (2 M tris(hydroxymethyl)aminomethane (Tris), 1 M acetic acid, 50 mM ethylenediaminetetraacetic acid (EDTA), pH 8.4) was purchased from Thermo Fischer Scientific. For the agarose gel electrophoresis, the ethidium bromide and gel loading dye solution were purchased from Sigma Aldrich, whereas the agarose was purchased from Meridian Bioscience. The gel loading dye solution (0.25% bromophenol blue, 0.25% xylene cyanol, 40% sucrose) was diluted 1:30 in 40% (w/v) sucrose before used. The uranyl formate for staining the TEM samples were purchased from Electron Microscopy Sciences. The citrate stabilized gold nanoparticles (10 nm in diameter) were purchased from either Sigma Aldrich or nanoComposix. For all of the experiments, deionized water (Milli-Q grade) was used.

DNA Origami Concentration Estimation
The DNA origami concentration after PEG purification was estimated from the DNA origami absorbance at 260 nm using Beer-Lambert law: where A 260 is the absorbance at a wavelength of 260 nm, ϵ 260 is the approximated molar extinction coefficient of the DNA origami structure at 260 nm and l is the path lenght through the solution in centimeters (0.05 cm in the used set-up). The absorbance at 260 nm was measured using a BioTek Eon Microplate Spectrophotometer and a Take3 microvolume plate. A sample size of 2 µL was used for the measurements and the concentration was obtained as the average of three measurements. The molar extinction extinction coefficients, ϵ 260 , where calculated as where N ds is the number of hybridized nucleotides and N ss is the number of non-hybridized, single-stranded nucleotides in the DNA origami unit.(1) DNA origami unit Extinction coefficient (M -1 cm -1 ) DNA origami unit with pH latches, polyT-passivated 1.02 × 10 8 Permanently closed DNA origami unit, polyT-passivated 1.02 × 10 8 Permanently open DNA origami unit, polyT-passivated 1.01 × 10 8 DNA origami unit with pH latches for dimer formation 1.01 × 10 8 DNA origami unit with pH latches, polyT-passivated on the top arm (used for 1D arrays) 0.98 × 10 8 DNA origami unit with pH latches and scaffold loops (used for 2D lattices) 0.94 × 10 8 DNA origami unit with pH latches, AuNP attachment strands and scaffold loops (used for 2D lattices) 0.95 × 10 8 Permanently closed DNA origami unit with scaffold loops (used for 2D lattices) 0.94 × 10 8 Permanently closed DNA origami unit with AuNP attachment strands and scaffold loops (used for 2D lattices) 0.94 × 10 8

AFM Imaging of the DNA Origami Unit
10 µL of poly-T passivated DNA origami units with pH latches (PEG-purified, c = 1 nM for the sample at pH 8.2 and c = 2 nM for the sample at pH 6.0) was deposited onto a freshly cleaved mica surface (15 mm × 15 mm, grade V1, Electron Microscopy Sciences) and incubated covered at room temperature for 1 min. After the incubation, the mica surface was rinsed 3 times with 100 µL of deionized water, after which the sample was dried thorouhly using a nitrogen gas stream. The atomic foce microscopy (AFM) images were obtained using a Dimension Icon AFM (Bruker). The samples were imaged in air using ScanAsyst in Air Mode and ScanAsyst-Air probes (Bruker). The AFM images were recorded with a resolution of 512 pxl × 512 pxl, a scan rate of 0.5 Hz and a scan size of 2 µm × 2 µm. The images were processed using Gwyddion open source software (v. 2.58). (2)

pH-Responsiveness of Rehydrated 2D DNA Origami Lattices
In order to demonstrate that assembled and dried 2D lattices still are pH-responsive and reconfigurable if rehydrated, the following approach was used. PEG-purified DNA origami units (final concentration of 2.0 nM) were mixed with 10-fold excess of connector oligonucleotides in 1× TAE supplemented with 12.5 mM MgCl 2 and 75 mM NaCl at pH 6.0 (lattice assembled at pH 6.0) or in 1× TAE supplemented with 10 mM MgCl 2 and 75 mM NaCl at pH 8.2 (lattice assembled at pH 8.2). 120 µL of the DNA origami sample mixture was evenly deposited onto a freshly cleaved mica surface (15 mm × 15 mm, grade V1, Electron Microscopy Sciences) and incubated covered at room temperature for 3 or 24 h. After the incubation, the mica surface was rinsed 3 times with 100 µL of deionized water, after which the sample was dried thoroughly using a nitrogen gas stream. Note that, the rehydrated lattice will not be pH-responsive and reconfigurable if attached to the mica substrate with NiCl 2 before the drying (see Figure S36).
After drying and AFM imaging of the sample, the lattice was rehydrated and at the same time the pH increased/decreased. In order to rehydrate (and increase the pH of) the lattice assembled at pH 6.0, 120 µL of 1× TAE, 20 mM MgCl 2 and 75 mM NaCl at pH 8.2 was deposited on the lattice and incubated covered for 2 hours. Similarly, in order to rehydrate (and decrease the pH of) the lattice assembled at pH 8.2, 120 µL of 1× TAE, 20 mM MgCl 2 and 75 mM NaCl at pH 6.0 was deposited on the lattice and incubated covered for 20 hours. After the incubation, the mica surface was rinsed 3 times with 100 µL of 1× TAE, 20 mM MgCl 2 at pH 8.2 (for the lattice changed to pH 8.2) or 100 µL of 1× TAE, 20 mM MgCl 2 at pH 6.0 (for the lattice changed to pH 6.0). Immediately after this washing step, 120 µL of 1× TAE, 10 mM NiCl 2 was deposited on the mica surface and incubated covered for 1 h. After the incubation, the mica surface was rinsed 6 times with 100 µL of deionized water, after which the sample was dried thoroughly using a nitrogen gas stream.
The atomic foce microscopy (AFM) images were obtained using a Dimension Icon AFM (Bruker). The samples were imaged in air using ScanAsyst in Air Mode and ScanAsyst-Air probes (Bruker). The AFM images were recorded with a resolution of 512 pxl × 512 pxl, a scan rate of 0.75 Hz and a scan size of 3 µm × 3 µm. The images were processed using Gwyddion open source software (v. 2.58). (2)                         The lattices are attached to the mica substrate with NiCl2 before drying and rehydration. Therefore, the lattices will not change its conformation even if the pH is increased.
The size of the images is 2 µm × 2 µm.

S50
All versions of the DNA origami unit contain the core staple strands listed in Table S2. Depending on the unit type and wanted functionality, some of the core staple strands in Table S3 could be replaced with staple strands in Table S4 (pH latches), Table S5 (sequences for permanently closing the unit), Table S6 (reverse pH latches) or Table S7 (AuNP attachment strands). The pH latches and the sequences for permanently closing the unit has been adapted from Ijäs et al. (3). The side strands with the poly-T 8 overhangs are listed in Table S8. For selectively connecting several units together, these side strands could be replaced with the connector oligonucleotides in Table S9. The replacement is always done for the whole set of staple strands of the same type, for example, the whole set "A of poly-T passivated side-strands" would always be replaced with the whole set "bottom arm, A".        ttttttttGCTAAACAACTTTCAATTCTGTAACGATCT Table S9. The sequences for connector oligonucleotides. The staple strands are written in the 5' to 3' direction and the Start -End location refers to the position in the caDNAno design. The 3-nt long overhangs (in the 3' end) complementary to the scaffold sequence on the opposite end of the arm are written in lowercase letters.