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Single-Molecule FRET & Super-Resolution

DNA Nanostructures Organize Dyes for Nanophotonic Apps

Jun. 20, 2011
Fig. 1: Sketch of single-molecule four-color spectroscopy of FRET pathways on DNA origami. DNA nanostructures are measured when diffusing through the laser focus. 10 photon streams are measured to identify subpopulations and quantify energy transfer.
Fig. 1: Sketch of single-molecule four-color spectroscopy of FRET pathways on DNA origami. DNA ... more
Fig. 1: Sketch of single-molecule four-color spectroscopy of FRET pathways on DNA origami. DNA ... Fig. 2: A) Scheme of the DNA origami technique: A long scaffold strand is folded into the desired ... Fig. 3: Histograms of FRET related ratios show how the FRET is shifted depending on the indicated ... Fig. 4: The new four-color technique allows selecting the population of interest in so-called ... Fig. 5: DNA nanostructures for use as nano-scale yardsticks for super-resolution microscopes. A) ... 

In order for nanotechnology, photonics and single-molecule spectroscopy to meet, structures with defined molecular compositions with dimensions in the 1-100 nm range are required. With the aid of DNA, nanostructures were constructed that guide light in switchable directions using multistep FRET from an input dye to an output dye. The direction of FRET is controlled by a jumper dye. At longer length-scales rigid DNA structures serve as standards for super-resolution microscopes.

Light Information Processing Beyond the Diffraction Limit

The wave nature of light and the associated diffraction not only undermine resolution of common light microscopes but also impose the difficulty of miniaturizing photonic devices such as fiber optic cables and couplers. In order for light-based circuitry to be competitive with current electronic circuits and to overcome the speed limitations of electronics, this size compatibility problem is a central challenge. Coupling the light into surface plasmons and using excited state interactions of molecules represent possible solutions. For plasmonic circuits, components such as wires, switches and connectors are required. Some of these tasks could be carried out by molecular devices such as photonic wires and molecular switches involving organic fluorescent dyes or quantum dots [1]. Fluorescence Resonance Energy Transfer (FRET) offers a means of excited state energy transport that is exploited in molecular photonic wires, devices that can be viewed as nano-scale optical waveguides [2]. Furthermore, functions such as optical switching can be achieved using the photochemistry of single chromophores [3].

Nanostructures for DNA Photonics

When molecular building blocks are used, a key question is how these building blocks can be arranged with respect to each other, since functioning of the molecular devices crucially depends on their arrangement.

In addition, the use of molecules in nano-scale devices implies that a very small number of molecules has to carry out the function with high reliability and minimal aging effects. A material that has the potential to act as a template for this exquisite organization of single molecules is DNA. From a materials perspective, DNA is a polymer that can be addressed over large distances along a double stranded helix exploiting the specific recognition that is provided by base pairing interactions. DNA nanotechnology meanwhile provides the tools to convert the control along the DNA into a true 3D spatial arrangement. Using a technique called DNA origami [4], hundreds of short synthetic DNA staple strands are hybridized to a long single-stranded DNA scaffold molecule (fig. 2A). Hybridization folds the scaffold into a two- or three-dimensional structure whose shape can be specified by the choice of the staple strand sequences. Using modified staple strands, carrying for example fluorescent dyes, allows their precise arrangement within the forming structures. The self-assembly yields billions of identical functional nanostructures.

We used the ability to arrange fluorophores with the aid of DNA at two length scales. Below 10 nm, we arranged fluorophores for multistep energy transfer in a photonic wire like device with switching functionality using an energy transfer cascade [1]. Alternatively, we inserted fluorescent dyes at larger distances of > 10 - 200 nm. The precise control over these length scales is a unique ability of comparably stiff DNA nanostructures and has enabled nanometer yardsticks to test super-resolution microscopes [5].

Energy Transfer Paths on DNA Origami

On a rectangular DNA origami, we engineered an energy transfer system based on single dye molecules with the functionality to control the energy transfer path. We used a "blue" fluorophore (ATTO488) as input dye and a "red" fluorophore (ATTO647N) as well as an "IR" fluorophore (Alexa 750) as alternative output dyes (fig. 2B). A "green" fluorophore (ATTO565) can be placed at two alternative locations and serves as jumper dye directing the excitation energy either to the red or to the IR dye (see fig. 2B, magnified view). Distances between input and output dyes are of the order of 9 nm to minimize direct FRET from input to output dye but to enable successive (hopping) FRET with the aid of the jumper dye.

Upon insertion of the jumper dye between the blue and the red dye, the FRET related ratio from blue to red increases from zero to 0.34 ± 0.12 indicating a two-step energy transfer from blue to red enabled by the green jumper dye (fig. 3). At the same time, the mean energy transfer between the blue and the IR dye is unchanged. Alternatively, the jumper dye was inserted between the blue and the IR dye. This causes the FRET related ratio to increase from the blue to the IR dye from zero to 0.25 ± 0.13 whereas the energy transfer from the blue to the red dye remains at zero.

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Keywords: DNA dSTORM Fluorescence Resonance Energy Transfer FRET. Super-Resolution Microscopy Ingo Stein Nanophotonics Nanostructures Nanotechnology Philip Tinnefeld photonics STED microscopy

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