Using an atomic force microscope as a “crane”, LMU (Ludwig-Maximilians-Universität München) researchers have succeeded in bringing two biomolecules together to form an active complex – with nanometer precision and built-in quality control.
The business end of the atomic force microscope (AFM) is its needle-sharp tip. It can be used to pick single molecules from a substrate and move them to specific positions with the precision of a few nanometers. This “single-molecule cut-and-paste” procedure was developed by LMU physicist Professor Hermann Gaub, and he and his colleagues have now used it to assemble a functional molecular complex from inactive, single-molecule building blocks.
They built the complex from two short strands of RNA, picking one from a depot with the AFM, and placing it close to the second strand deposited elsewhere on the substrate. When the two RNA segments come into contact, they spontaneously form what is called an “aptamer”, a three-dimensional binding pocket for a target molecule – in this case the fluorescent dye malachite green. The binding interaction amplifies the fluorescence emitted by the target more than 1000-fold – and signals that the two parts of the aptamer have assembled correctly.
Mechanically assembled molecule
This is precursor work to more advanced mechanical nanotechnology where atoms are placed for atomically precise reactions. Here the placement is about one hundred times less precise than what is needed for atomically precise reactions.
Bottom up assembly of functional molecular ensembles with novel properties emerging from composition and arrangement of its constituents is a prime goal of nanotechnology. By single-molecule cut-and-paste we assembled binding sites for malachite green in a molecule-by-molecule assembly process from the two halves of a split aptamer. We show that only a perfectly joined binding site immobilizes the fluorophore and enhances the fluorescence quantum yield by several orders of magnitude. To corroborate the robustness of this approach we produced a micrometer-sized structure consisting of more than 500 reconstituted binding sites. To the best of our knowledge, this is the first demonstration of one by one bottom up functional biomolecular assembly.
“The important thing is that we have precise mechanical control over the assembly process,” says lead author Mathias Strackharn. “When we see the malachite-green signal in the fluorescence microscope, we know that the aptamer has been successfully reconstituted.” The researchers are now in a position to construct other systems whose natural function depends on the configuration of their molecular components. This will enable them to dissect how interactions between their parts mediate the functions of molecular complexes.
The positions of the Cy5 label (a), and the malachite green fluorescence bursts (b, c, and d in
temporal order) were determined by fitting gaussians to the diffraction limited spots. The
diffraction limited fluorescent pattern depicted in this graph is that of the malachite green
fluorescence burst of (d). Error bars represent the fitting errors. Deviations from the original Cy5
position are on the order of 10 nm and can be assigned to thermal drift.