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,
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.source: nanoweb.org,written by Belle Dumé i
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