Programmable Multivalent DNA-Origami Tension Probes for Reporting Cellular Traction Forces.

Mechanical forces are central to most, if not all, biological processes, including cell development, immune recognition, and metastasis. Because the cellular machinery mediating mechano-sensing and force generation is dependent on the nanoscale organization and geometry of protein assemblies, a current need in the field is the development of force-sensing probes that can be customized at the nanometer-length scale. In this work, we describe a DNA origami tension sensor that maps the piconewton (pN) forces generated by living cells. As a proof-of-concept, we engineered a novel library of six-helix-bundle DNA-origami tension probes (DOTPs) with a tailorable number of tension-reporting hairpins (each with their own tunable tension response threshold) and a tunable number of cell-receptor ligands. We used single-molecule force spectroscopy to determine the probes' tension response thresholds and used computational modeling to show that hairpin unfolding is semi-cooperative and orientation-dependent. Finally, we use our DOTP library to map the forces applied by human blood platelets during initial adhesion and activation. We find that the total tension signal exhibited by platelets on DOTP-functionalized surfaces increases with the number of ligands per DOTP, likely due to increased total ligand density, and decreases exponentially with the DOTP's force-response threshold. This work opens the door to applications for understanding and regulating biophysical processes involving cooperativity and multivalency.

Reconfigurable Three-Dimensional Gold Nanorod Plasmonic Nanostructures Organized on DNA Origami Tripod.

Distinct electromagnetic properties can emerge from the three-dimensional (3D) configuration of a plasmonic nanostructure. Furthermore, the reconfiguration of a dynamic plasmonic nanostructure, driven by physical or chemical stimuli, may generate a tailored plasmonic response. In this work, we constructed a 3D reconfigurable plasmonic nanostructure with controllable, reversible conformational transformation using bottom-up DNA self-assembly. Three gold nanorods (AuNRs) were positioned onto a reconfigurable DNA origami tripod. The internanorod angle and distance were precisely tuned through operating the origami tripod by toehold-mediated strand displacement. The transduction of conformational change manifested into a controlled shift of the plasmonic resonance peak, which was studied by dark-field microscopy, and agrees well with electrodynamic calculations. This new 3D plasmonic nanostructure not only provides a method to study the plasmonic resonance of AuNRs at prescribed 3D conformations but also demonstrates that DNA origami can serve as a general self-assembly platform for constructing various 3D reconfigurable plasmonic nanostructures with customized optical properties.

Plasmonic Toroidal Metamolecules Assembled by DNA Origami.

We show hierarchical assembly of plasmonic toroidal metamolecules that exhibit tailored optical activity in the visible spectral range. Each metamolecule consists of four identical origami-templated helical building blocks. Such toroidal metamolecules show a stronger chiroptical response than monomers and dimers of the helical building blocks. Enantiomers of the plasmonic structures yield opposite circular dichroism spectra. Experimental results agree well with the theoretical simulations. We also show that given the circular symmetry of the structures s distinct chiroptical response along their axial orientation can be uncovered via simple spin-coating of the metamolecules on substrates. Our work provides a new strategy to create plasmonic chiral platforms with sophisticated nanoscale architectures for potential applications such as chiral sensing using chemically based assembly systems.

A DNA-directed light-harvesting/reaction center system.

A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.

Reengineering the optical absorption cross-section of photosynthetic reaction centers.

Engineered cysteine residues near the primary electron donor (P) of the reaction center from the purple photosynthetic bacterium Rhodobacter sphaeroides were covalently conjugated to each of several dye molecules in order to explore the geometric design and spectral requirements for energy transfer between an artificial antenna system and the reaction center. An average of 2.5 fluorescent dye molecules were attached at specific locations near P. The enhanced absorbance cross-section afforded by conjugation of Alexa Fluor 660 dyes resulted in a 2.2-fold increase in the formation of reaction center charge-separated state upon intensity-limited excitation at 650 nm. The effective increase in absorbance cross-section resulting from the conjugation of two other dyes, Alexa Fluor 647 and Alexa Fluor 750, was also investigated. The key parameters that dictate the efficiency of dye-to-reaction center energy transfer and subsequent charge separation were examined using both steady-state and time-resolved fluorescence spectroscopy as well as transient absorbance spectroscopy techniques. An understanding of these parameters is an important first step toward developing more complex model light-harvesting systems integrated with reaction centers.

DNA-directed artificial light-harvesting antenna.

Designing and constructing multichromophoric, artificial light-harvesting antennas with controlled interchromophore distances, orientations, and defined donor-acceptor ratios to facilitate efficient unidirectional energy transfer is extremely challenging. Here, we demonstrate the assembly of a series of structurally well-defined artificial light-harvesting triads based on the principles of structural DNA nanotechnology. DNA nanotechnology offers addressable scaffolds for the organization of various functional molecules with nanometer scale spatial resolution. The triads are organized by a self-assembled seven-helix DNA bundle (7HB) into cyclic arrays of three distinct chromophores, reminiscent of natural photosynthetic systems. The scaffold accommodates a primary donor array (Py), secondary donor array (Cy3) and an acceptor (AF) with defined interchromophore distances. Steady-state fluorescence analyses of the triads revealed an efficient, stepwise funneling of the excitation energy from the primary donor array to the acceptor core through the intermediate donor. The efficiency of excitation energy transfer and the light-harvesting ability (antenna effect) of the triads was greatly affected by the relative ratio of the primary to the intermediate donors, as well as on the interchromophore distance. Time-resolved fluorescence analyses by time-correlated single-photon counting (TCSPC) and streak camera techniques further confirmed the cascading energy transfer processes on the picosecond time scale. Our results clearly show that DNA nanoscaffolds are promising templates for the design of artificial photonic antennas with structural characteristics that are ideal for the efficient harvesting and transport of energy.