Jun 27, 2014
DNA chimeras help assemble protein nanodevices
Most nanoscale components today are made by top-down processes, like lithography, where smaller structures are created from a larger starting block. Such techniques are routinely employed in the semiconductor industry but the goal of nanotechnology is to build tiny devices from the bottom up, which is much more challenging. Researchers in Germany have now shown that their "single-molecule cut-and-paste" technique can be used to make protein-DNA chimeras for such nano-assembly and to study the fundamental behaviour of biomolecules such as proteins.
In 2008, Hermann Gaub and colleagues at the University of Munich developed a single-molecule cut-and-paste (SMC&P) technique that involves using an atomic force microscope (AFM) tip to pick up "sticky" DNA molecules, which bind only selectively to certain other complementary DNA molecules. The AFM works rather like a “nanocrane” that lifts up biomolecules, just like a normal, everyday crane lifts up life-sized objects. The molecules can then be brought to a target “construction site”, where they may be arranged at will.
In recent years, the researchers have considerably improved their method and can now even use it to transport DNA-coupled proteins, which are complex molecules. Such protein-DNA chimeras, as they are known, are also very useful in immunobiology applications as well as in nanobiotechnology – in particular for so-called DNA origami.
Gaub’s team has now taken its technique even further by employing the 11 amino acid ybbR-tag, helped along by the enzyme phospopantetheinyl transferase Sfp, to selectively attach co-enzyme A-modified DNA to proteins (see figure).
Transporting single protein molecules
“By means of the ybbR-tag/CoA/Sfp system, we can efficiently and specifically couple proteins and DNA, and obtain robust protein-DNA chimeras that can be used in SMC&P,” says team member Diana Pippig. “Since the protein-DNA chimeras are so well defined and behaved we have succeeded in efficiently and precisely transporting actual single protein molecules, something hitherto only possible for mere DNA molecules.”
The researchers began by assembling the transfer complex comprising a protein (in this case “GFP”) and a covalently attached short-DNA-transfer strand. “We genetically modified the GFP to harbour encoded short peptide tags (an 11 amino acid ybbR-tag at one end and a 12 amino acid ‘GCN4-tag’ at the other),” explains Pippig. “We also modified the DNA oligomer with a co-enzyme A group so that it selectively attaches to the protein.”
What happens next is that the phospopantetheinyl transferase Sfp acts as a catalyst to couple the co-enzyme A to the hydroxyl group of a Serine residue in the ybbR-tag. “We then store the transfer complex in a predefined depot area on a functionalized glass surface by hybridizing the complex with a surface-bound, complementary DNA oligomer,” Pippig told nanotechweb.org.
Picking up and pasting
The “cut” part of the process then involves the DNA molecule being unzipped at its bound end. The GFP-DNA complex is picked up by the AFM-tip transport crane and pasted in the depot area. In fact, the AFM tip contains a single-chain antibody fragment that specifically binds to the GCN4 peptide tag in the protein.
“In each SMC&P transfer cycle, we pick up one of the protein-DNA complexes stored in the depot area using the AFM tip and then deposit it where we want in the target area,” says Pippig. “The antibody AFM tip then becomes free again to move to the depot area and pick up a new transfer complex.”
Our process is precise to a scale of 10 nm, and we can pick up and arrange proteins from an arbitrary number of spatially separated depot regions, by, for example, employing a multichannel microfluidic device, she adds. “Indeed, such precision should enable us to position a protein in a confined compartment, such as a ‘zero-mode’ waveguide, for instance, which would allow us to observe single-molecule protein reactions using fluorescence spectroscopy. And that is not all: we might even be able to place proteins close to one another in a target area and study how they interact.”
The team describes its work in ACS Nano DOI: 10.1021/nn501644w.
About the author
Belle Dumé is contributing editor at nanotechweb.org
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