Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching

Abstract

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.

Towards control of excitonic coupling in DNA-templated Cy5 aggregates: the principal role of chemical substituent hydrophobicity and steric interactions

“…18,19 Importantly, due to the programmable nature of DNA nanotechnology and the nm scale of the DNA double helix, the positioning of dyes can be controlled down to the single nm level. 20,21 By placing multiple dyes within a small area of the DNA scaffold, precise aggregates can be formed at low concentrations, which is something particularly challenging when dealing with both highly soluble or highly insoluble dyes. While computational chemistry offers great insight, 22 the varying factors that regulate coupling such as energy levels, transition dipole moments (TDM), electrostatics, hydrophobicity, etc., are hard to capture without experimental data to support the models.…”

“…Importantly, they are also readily available as labels of oligonucleotides from commercial vendors. 20 The red-shifted indodicarbocyanines (Cy5, having a five methine bridge) have been a recent focus because of the large TDM (the absolute square of the TDM being proportional to the measured molar absorptivity, ∼250 000 M −1 cm −1 at peak), which contributes to producing strong coupling. [23][24][25] Additionally, their absorption and fluorescence spectra are in the visible light range, allowing for detailed characterization of monomers and aggregates on common spectroscopic systems.…”

Prediction and Control in DNA Nanotechnology

“…Overall, AAMD is generally not used for simulating entire DNA origami devices. The computational cost of atomistic simulations often restricts their use to studying small DNA origami constructs (such as Seeman J1 sequences , ), subsections of DNA devices (where the rest of the DNA origami is fixed ,, ), or large structures for very short times …”

“…This enables time scale access approaching that of AAMD while still capturing the physics required for phenomena such as ATP hydrolysis or enzyme activity . In the context of DNA nanotechnology, combinations of QM and MD simulations have already been applied toward the optimization of dye placement to afford control over device signal output and could find future use in photonic systems or systems containing quantum dots. − …”

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards

“…104 Even more interesting is that it may also be able to help address the challenge of orienting the dyes. 105,106 They showed how single Cy3 and Cy5 dye molecules could be incorporated in a DNA origami nanostructure with controlled orientation by doubly linking them to oligonucleotide strands that were hybridized while leaving key bases unpaired in the scaffold. Increasing the number of bases unpaired induced a stretching of the fluorophore linkers and reduced its freedom of mobility, which left more space for the fluorophore to accommodate and find different sites for interaction with the DNA.…”