A team of researchers from Aarhus University (Aarhus, Denmark) has utilized atomic force microscopy (AFM) to observe single molecules as they attach to a DNA origami scaffold. DNA origami—the nanoscale folding of DNA to create arbitrary two- and three-dimensional shapes—was developed by Paul Rothemund of the California Institute of Technology in 2006 (1). AFM, which was first introduced by G. Binnig and C.F. Quate of Stanford University, and C. Gerber at the IBM San Jose Research Laboratory in 1986 (2), uses a high-resolution, scanning probe microscope that can capture images at a resolution as small as fractions of a nanometer. The new research, led by organic chemist Kurt Gothelf, generated a protocol for attaching single molecules to DNA origami scaffolds by observing chemical reactions between the molecules with AFM.
Although the use of AFM to analyze DNA origami shapes has been previously described, researchers have not been able to use AFM to view the chemical reactions that enable single molecules to bind with the DNA origami scaffold because the molecules are too small to appear clearly. “Unless small molecules are organized in lattice structures on surfaces it very difficult to image and identify them by AFM,” Gothelf told BioTechniques. The researchers therefore devised a new way to increase the visibility of single molecules so that AFM could capture the reactions.
Gothelf said that the team made two key changes to the protocol for imaging with AFM that made it possible to view single molecules on the surface of a DNA origami scaffold. First, the researchers attached the small molecule biotin to all groups of molecules that were either cleaved or bound to the surface of the DNA origami strand. Then, they introduced the large protein streptavidin, which binds strongly with biotin. AFM was able to detect the larger streptavidin-biotin complex created by the researchers. “In the AFM images even streptavidin appears only as bright dots on the surface, and if they were distributed randomly at the surface, it would be very difficult to gain any information from their appearance or disappearance,” said Gothelf. “However, by using the DNA origami we know the streptavidin is supposed to be at the surface, and therefore we can derive precise information about the chemical reactions.”
The team tested this method on a two-dimensional DNA origami structure formed by folding a single-stranded piece of DNA from the bacteriophage M13mp18. Oligonucleotides tagged with the streptavidin-biotin complex were introduced to 12 locations on the DNA origami structure and were allowed to self-assemble. The researchers reported 84% efficiency for identifying the exact location of the tagged oligonucleotides.
According to Gothelf, it is important to be able to analyze molecular interactions as the DNA origami structure assembles on a nanoscale level because it will help researchers manipulate these bonds. “It is very difficult to control matter at the nanoscale,” said Gothelf. “Therefore, it is important to develop methods that mimic natural self-assembly. Nature’s machinery is constructed by making covalent chemical bonds, and here we show for the first time that it is possible to control and image the formation of covalent chemical bonds in complex artificial systems, and this is an important step towards creating artificial devices.” Such devices could be used for optical or electronic applications.
The origami structure used for this research contained more than 200 pixel positions that could support a binding event with a single oligonucleotide, Gothelf said. These positions were located approximately 6 nm apart. The researchers new protocol allowed them to image the chemical reactions occurring at the individual pixel locations. “We are now pursuing connections between the pixels, by connecting organic molecules,” said Gothelf. “This might make us able to make small circuits in the origami surfaces, which would be useful for the integration of biomolecular sensing in circuitry.”
The paper, “Small molecule induced control in duplex and triplex DNA-directed chemical reactions,” was published in the March issue of Nature Nanotechnlogy.
References:
1. Paul W.K. Rothmund "Folding DNA to create nanoscale shapes and patterns" Nature 440 297-303 March 16, 2006
2. G. Binnig, C.F. Quate, C. Gerber "Atomic Force Microscope" Physical Review Letters 56:9 1986