Folding and Characterization of a Bio-responsive Robot from DNA Origami

Summary

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

Abstract

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

Introduction

The uses for nucleic acids nanotechnology are astounding. The tractability of the Watson-Crick base pairing as well as the ease and relative low-cost of large-scale synthesis of custom-made oligos² has generated an explosion of applications³ and research in the field of DNA nanotechnology. Structural DNA nanotechnology, based on the immobile Seeman junction^4,5 as a fundamental building block makes use of DNA as a self-assembling elementary unit for the construction of arbitrary shapes^6-8.

The recent development of the scaffolded DNA origami⁹ technique allows for the construction of comp....

Protocol

1. Preparation of Staples Pool Mixture

1. Order lyophilized DNA nanorobot staples on 96-well plates as listed in Table 1 (see Materials) and normalize to 10 nmol. For a detailed description of the design and architecture of the DNA nanorobot see Ben-Ishay et al.¹⁴ and Douglas et al.¹⁵).
2. Reconstitute each staple well with DNase/RNase-free ultrapure to a concentration of 100 µM. For staples normalized to 10 nmol, reconstitute with 100 µl of ultrapure water.
3. Pool together 20 µl of each staple using a multichannel pipettor and a sterile 55 ml solution basi....

Results

Representative results are shown in Figure 2A. All lanes contain 1 µg of total DNA, measured via spectrophotometer (OD₂₆₀). Compared with the circular single-strand DNA scaffold (Lane 2), nanorobots are hindered in the gel due to their higher molecular weight, the result of staples hybridization to the scaffold DNA (Lane 3. Red arrow). The low molecular weight band in Lane 3 represents excess staples which did not bind to the scaffold DNA (Green arrow). After purification via centrifugal filtr.......

Discussion

We described the fabrication, purification, and visualization of the DNA nanorobot. Following fabrication of the hexagonal chassis of the device, the function of the nanorobot is programmed with the simple introduction of specific cargo and sensing strands to the robot which readily find their designated position due to hydrogen-bonding complementarity with available single-strand docking sites^14,15,22.

The fabrication protocol described uses a slow annealing ramp, which is generall.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DNase/RNase free distilled water	Gibco	10977
M13mp18 ssDNA scaffold	NEB	N4040S
10x TAE	Gibco	15558-042
1 M MgCl2	Ambion	AM9530G
Amicon Ultra 0.5 ml centrifugal filter 100K MWCO	Amicon	UFC510024
Agarose	Promega	V3125
TBE buffer	Promega	V4251
Ethidium bromide 10 mg/ml solution	Sigma Aldrich	E1510
1 kb DNA marker	NEB	N3232S
Loading Dye	NEB	B7021S
uranyl formate	polysciences	24762
carbon-coated TEM grids	Science services	EFCF400-Cu-50
Thermal Cycler c1000 Touch	Bio-Rad
Glow Discharge K100X	Emitech
UV table Gel Doc EZ Imager	Bio-Rad
NanoDrop 2000c	Thermo Scientific
TEM FEI-G12	Tecnai