Single-Molecule FRET & Super-Resolution
DNA Nanostructures Organize Dyes for Nanophotonic Apps
- 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. 2: A) Scheme of the DNA origami technique: A long scaffold strand is folded into the desired strand using ~ 200 short staple strands. Modifications such as dyes are easily incorporated. B) Arrangement of fluorophores on the DNA origami and visualization of alternative energy-transfer pathways with the “jumper” dye guiding the light from the blue input to either the red or the IR output.
- Fig. 3: Histograms of FRET related ratios show how the FRET is shifted depending on the indicated position of the jumper dye.
- Fig. 4: The new four-color technique allows selecting the population of interest in so-called stoichiometry histograms. The compositions of the identified populations are indicated by colored circles.
- Fig. 5: DNA nanostructures for use as nano-scale yardsticks for super-resolution microscopes. A) Comparison of diffraction limited and super-resolved images of two dyes placed at a distance of 60 nm. B) Histogram of single-molecule localizations along the horizontal of the super-resolved image. C) Sketch of the nano-scale yardstick and the principle of DNA-PAINT. D) Distribution of measured distances for many individual structures.
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 . 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 . Furthermore, functions such as optical switching can be achieved using the photochemistry of single chromophores .
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 , 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 . 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 .
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.
Single-Molecule Four-Color FRET
Due to the complexity of these supra-molecular constructs we expected considerable sample heterogeneity. This heterogeneity is best addressed using single-molecule techniques. For the FRET results reported above, a new single-molecule four-color FRET technique with alternating laser excitation that allows studying the interaction between six energy transfer pairs quasi-simultaneously was developed. The setup is based on an inverted confocal microscope and uses the combination of a supercontinuum laser for free wavelength selection with acousto-optical filters (AOTFs) and beamsplitter (AOBS) for excitation alternation and optimized detection sensitivity on all channels. Alternating laser excitation enables direct probing of the stoichiometry by subsequently exciting all four fluorophores. The different subpopulations are evaluated by the relative intensities in so-called stoichiometry-histograms (fig. 4).
In the two-dimensional histogram comparing all four dyes in the construct, the population around 0.5 on both axes represents intact DNA origami with all four fluorophores. Interestingly, detailed analysis revealed that origami structures were formed with very high yield and sample heterogeneity mainly originated from inactive subpopulations of the IR-dye. Energy transfer was analyzed one molecule at a time and the results were plotted in histograms (fig. 3). Detailed analysis revealed that for the selected thresholds for > 98% of the molecules, the position of the jumper dye could be correctly assigned.
Yardsticks for Super-Resolution Microscopy
Positioning dyes at distances too large for FRET to occur is the key for one of the first practical applications of DNA nanostructures, that is, DNA nano-scale yardsticks for super-resolution microscopy.
The common principle of super-resolution microscopy by subsequent single-molecule localizations is that only one fluorophore is active for a diffraction-limited area at any given time. Every fluorophore is localized by imaging with a sensitive camera and the molecules' positions are summed up in histograms to obtain the reconstructed super-resolved images.
To compare super-resolution techniques and to test super-resolution microscopes, we developed microscopy standards with an exactly defined number of fluorophores at programmed distances in the range of 10-200 nm prepared by the DNA origami technique . We used different super-resolution techniques such as Blink-Microscopy  which exploits radical ion dark states, STED, DNA-PAINT , and dSTORM  to resolve distances between single molecules or lines of molecules beyond the diffraction limit. In figure 5, an example of two dyes separated by 60 nm resolved by DNA-PAINT is depicted. These nanoscopic yardsticks can be easily distributed and might become an important alignment tool for super-resolution microscopes.
In summary, the combination of DNA as a template with photonic functionalities placed on top is becoming an emerging field of research and might have the potential to fill the gap between top-down lithography and bottom-up self-assembly.
The authors are grateful to Sebastian Laurien and Christian Steinhauer for their help with the manuscript and figures. The work was supported by the DFG and the excellence cluster Nanosystems Initiative Munich (NIM).
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Prof. Dr. Philip Tinnefeld (corresponding author)
Institute of physical and theoretical Chemistry, Braunschweig University of Technology, Germany
& Center for NanoScience, Munich, Germany
Applied Physics - Biophysics & Center for NanoScience
Ludwig Maximilian University, Munich, Germany