A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns

The clustering of death receptors (DRs) at the membrane leads to apoptosis. With the goal of treating tumours, multivalent molecular tools that initiate this mechanism have been developed. However, DRs are also ubiquitously expressed in healthy tissue. Here we present a stimuli-responsive robotic switch nanodevice that can autonomously and selectively turn on the display of cytotoxic ligand patterns in tumour microenvironments. We demonstrate a switchable DNA origami that normally hides six ligands but displays them as a hexagonal pattern 10 nm in diameter once under higher acidity. This can effectively cluster DRs and trigger apoptosis of human breast cancer cells at pH 6.5 while remaining inert at pH 7.4. When administered to mice bearing human breast cancer xenografts, this nanodevice decreased tumour growth by up to 70%. The data demonstrate the feasibility and opportunities for developing ligand pattern switches as a path for targeted treatment.


Mean structure from oxDNA simulation
Supplementary Fig. 2: The mean origami structure computed from oxDNA simulation.Structures at the up panel are the screenshots of different views in oxView.Structures at the bottom panel are the re-rendered mean structure for better visualization, which was achieved by converting the mean structure into its pdb format first, and then the pdb file of the structure was rendered as its surface model.

Calculation consideration on distance from peptide to origami surface
To determine whether the terminus of each TFO strand is inside or outside the cavity of DNA origami, their distances to the boundary plane of the cavity are calculated.We if Axi + Byi + Czi + D = 0 Finally, we defined the signed distance di as the product of the distance and the sign of (Axi + Byi + Czi + D).Taking into consideration of the peptide size Furthermore, to make the estimation closer to the reality, we took the von Der Waals diameter of the peptide (2.8 nm) into consideration.To calculate the distance correction due to the size of the peptide, we first found out the backbone direction of the terminus nucleotide which directly connected to the peptide.It happens to be the a3 vector of the nucleotide in oxDNA configuration file.Then we obtained the angle θ between the a3 vector and the normal vector of the boundary plane N. Finally, based on the geometry as shown in Fig. SY, the corresponding corrected distance can be obtained with the formula: d' = d -R (1 -cos θ) where d' is the corrected distance between the peptide to the boundary plane, d is the distance between the terminus of the TFO to the boundary plane, R is the radius of the peptide, which is 1.4 nm, θ is the angle between N and a3.Supporting Fig. 3: The peptide size and orientation information of the base that connects the peptide are included for the distance calculation.

Distances from the peptide to the origami surface
Supplementary Fig. 4: Real-time distances, from the TFO terminus (dots in orange) or from the peptide surface to the origami surface (dots in magenta, recalculated from the dataset of orange points via including the orientation information of the TFO terminus and the physical diameter of the peptide), along the simulation.

Negative-stain TEM image of the origami before UV irradiation (buffer exchanged from 5 mM MgCl2 into 1X PBS)
Supplementary Fig. 8: Negative-stain TEM image of the origami (buffer exchanged from the buffer containing 5 mM MgCl2, 5 mM TRIS, and 1 mM EDTA to 1X PBS, then kept at room temperature overnight) before UV irradiation.The scale bar stands for 100 nm.

Mini-Scaffold quantification of the UV-crosslinked origami
Supplementary Fig. 12: Quantification of mini-scaffold in origami.Origami structures that contain 1, 2, 3, 4, 5, or 6 mini-Scaffold were prepared and purified.Then they were incubated with a Cy5-labeled oligo that detects the mini-Scaffold.After purification, the same amounts of origami samples were loaded onto a 2% agarose gel (contains 0.5 mg mL -1 ethidium bromide and 10 mM MgCl2) for electrophoresis (in 0.5X TBE supplemented with 10 mM MgCl2, 90 volts for 2 hours).The gel was imaged under a UV channel (a, top) and a fluorescent channel (a, bottom).The Cy5 signal of each origami sample was also quantitatively measured using a multimode microplate reader (b).n = 3 independent measurements.Data are presented as mean ± standard deviation.

Cell interaction of the origami
Supplementary Fig. 18: Folds of origami associated with SK-BR-3 cells.This is measured using flow cytometry to measure the Cy5 fluorescent signal on cells.Before the measurement, cells were incubated with three different concentrations (1 nM, 5 nM, and 10 nM) of empty origami or origami switch (n = 3 independent measurements, data are presented as mean ± standard deviation).