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Nanoarchitectural control of matter is crucial for next-generation technologies. DNA origami templates are harnessed to accurately position single molecules; however, direct single molecule evidence is lacking regarding how well DNA origami can control the orientation of such molecules in three-dimensional space, as well as the factors affecting control. Here, we present two strategies for controlling the polar (θ) and in-plane azimuthal (ϕ) angular orientations of cyanine Cy5 single molecules tethered on rationally-designed DNA origami templates that are physically adsorbed (physisorbed) on glass substrates. By using dipolar imaging to evaluate Cy5′s orientation and super-resolution microscopy, the absolute spatial orientation of Cy5 is calculated relative to the DNA template. The sequence-dependent partial intercalation of Cy5 is discovered and supported theoretically using density functional theory and molecular dynamics simulations, and it is harnessed as our first strategy to achieve θ control for a full revolution with dispersion as small as ±4.5°. In our second strategy, ϕ control is achieved by mechanically stretching the Cy5 from its two tethers, being the dispersion ±10.3° for full stretching. These results can in principle be applied to any single molecule, expanding in this way the capabilities of DNA as a functional templating material for single-molecule orientation control. The experimental and modeling insights provided herein will help engineer similar self-assembling molecular systems based on polymers, such as RNA and proteins.
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Dense Super-Resolution Imaging of Molecular Orientation Via Joint Sparse Basis Deconvolution and Spatial Pooling
In single-molecule super-resolution microscopy, engineered point-spread functions (PSFs) are designed to efficiently encode new molecular properties, such as 3D orientation, into complex spatial features captured by a camera. To fully benefit from their optimality, algorithms must estimate multi-dimensional parameters such as molecular position and orientation in the presence of PSF overlap and model-experiment mismatches. Here, we present a novel joint sparse deconvolution algorithm based on the decomposition of fluorescence images into six basis images that characterize molecular orientation. The proposed algorithm exploits a group-sparsity structure across these basis images and applies a pooling strategy on corresponding spatial features for robust simultaneous estimates of the number, brightness, 2D position, and 3D orientation of fluorescent molecules. We demonstrate this method by imaging DNA transiently labeled with the intercalating dye YOYO-1. Imaging the position and orientation of each molecule reveals orientational order and disorder within DNA with nanoscale spatial precision.
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- Award ID(s):
- 1653777
- PAR ID:
- 10104459
- Date Published:
- Journal Name:
- 2019 IEEE 16th International Symposium on Biomedical Imaging (ISBI 2019)
- Page Range / eLocation ID:
- 325 - 329
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1109/isbi.2019.8759444
