Determining the In-Plane Orientation and Binding Mode of Single Fluorescent Dyes in DNA Origami Structures

Kristina Hübner, Himanshu Joshi, Aleksei Aksimentiev, Fernando D. Stefani, Philip Tinnefeld, and Guillermo P. Acuna
ACS Nano 15(3) 5109-5117 (2021)
DOI:https://doi.org/10.1021/acsnano.0c10259  BibTex

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We present a technique to determine the orientation of single fluorophores attached to DNA origami structures based on two measurements. First, the orientation of the absorption transition dipole of the molecule is determined through a polarization-resolved excitation measurement. Second, the orientation of the DNA origami structure is obtained from a DNA-PAINT nanoscopy measurement. Both measurements are performed consecutively on a fluorescence wide-field microscope. We employed this approach to study the orientation of single ATTO 647N, ATTO 643, and Cy5 fluorophores covalently attached to a 2D rectangular DNA origami structure with different nanoenvironments, achieved by changing both the fluorophores’ binding position and immediate vicinity. Our results show that when fluorophores are incorporated with additional space, for example, by omitting nucleotides in an elsewise doublestranded environment, they tend to stick to the DNA and to adopt a preferred orientation that depends more on the specific molecular environment than on the fluorophore type. With the aid of all-atom molecular dynamics simulations, we rationalized our observations and provide insight into the fluorophores’ probable binding modes. We believe this work constitutes an important step toward manipulating the orientation of single fluorophores in DNA origami structures, which is vital for the development of more efficient and reproducible self-assembled nanophotonic devices.

Abstract

We present a technique to determine the orientation of single fluorophores attached to DNA origami structures based on two measurements. First, the orientation of the absorption transition dipole of the molecule is determined through a polarization-resolved excitation measurement. Second, the orientation of the DNA origami structure is obtained from a DNA-PAINT nanoscopy measurement. Both measurements are performed consecutively on a fluorescence wide-field microscope. We employed this approach to study the orientation of single ATTO 647N, ATTO 643, and Cy5 fluorophores covalently attached to a 2D rectangular DNA origami structure with different nanoenvironments, achieved by changing both the fluorophores’ binding position and immediate vicinity. Our results show that when fluorophores are incorporated with additional space, for example, by omitting nucleotides in an elsewise doublestranded environment, they tend to stick to the DNA and to adopt a preferred orientation that depends more on the specific molecular environment than on the fluorophore type. With the aid of all-atom molecular dynamics simulations, we rationalized our observations and provide insight into the fluorophores’ probable binding modes. We believe this work constitutes an important step toward manipulating the orientation of single fluorophores in DNA origami structures, which is vital for the development of more efficient and reproducible self-assembled nanophotonic devices. 

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1 µs long MD simulation trajectories (two independent runs) of the ATTO 647N dye-conjugated to the DNA backbone in sample 1 having two unpaired bases near the dye in the DNA origami design. The scaffold strand of the DNA origami structure is shown in cyan, the staple strand carrying the dye molecule is shown in red and the other staple strand is shown in orange. The atoms of the dye molecule are shown using green spheres whereas the atoms of C6 molecules (anchor between the dye and DNA) are shown using magenta spheres. Water and counter ions are not shown for clarity.

1 µs long MD simulation trajectories (two independent runs) of the ATTO 647N dye-conjugated to the backbone of DNA systems in sample 2, having no unpaired bases in the DNA origiami. The scaffold strand of the DNA origami structure is shown in cyan, the staple strand carrying the dye molecule is shown in red and the other staple strand is shown in orange. The atoms of the dye molecule are shown using green spheres whereas the atoms of C6 molecules (anchor between the dye and DNA) are shown using magenta spheres. Water and counter ions are not shown for clarity.

1 µs long MD simulation trajectories (two independent runs) of the ATTO 647N dye-conjugated to a DNA base in sample 3, having no unpaired bases in the DNA origiami. The scaffold strand of the DNA origami structure is shown in cyan, the staple strand carrying the dye molecule is shown in red and the other staple strand is shown in orange. The atoms of the dye molecule are shown using green spheres whereas the atoms of C6 molecules (anchor between the dye and DNA) are shown using magenta spheres. Water and counter ions are not shown for clarity.

1 µs long MD simulation trajectories (two independent runs) of the Cy5 dye-conjugated to a DNA base in sample 1, having two unpaired bases near dye in the DNA origiami. The scaffold strand of the DNA origami structure is shown in cyan, the staple strand carrying the dye molecule is shown in red and the other staple strand is shown in orange. The atoms of the dye molecule are shown using green spheres whereas the atoms of C6 molecules (anchor between the dye and DNA) are shown using magenta spheres. Water and, counter ions are not shown for clarity.

1 µs long MD simulation trajectories (two independent runs) of the Cy5 dye-conjugated to a DNA base in sample 2, having no unpaired bases in the DNA origiami. The scaffold strand of the DNA origami structure is shown in cyan, the staple strand carrying the dye molecule is shown in red and the other staple strand is shown in orange. The atoms of the dye molecule are shown using green spheres whereas the atoms of C6 molecules (anchor between the dye and DNA) are shown using magenta spheres. Water and, counter ions are not shown for clarity.