Susan Buckhout-White

Exploring attachment chemistry with FRET in hybrid quantum dot dye-labeled DNA dendrimer composites

Luminescent semiconductor quantum dots (QDs) and a range of biomolecules are now being routinely co-integrated into functional optical devices in pursuit of creating novel ‘value added’ photonic and energy harvesting/transfer materials. Amongst the biological molecules, structural DNA architectures are particularly useful due to their unrivaled ability to assume almost any desired shape along with allowing fluorophores to be precisely arranged on them with controlled stoichiometry and sub-nanometer positional accuracy. The unique properties available to joint QD–DNA composites suggest them for a host of new applications in light harvesting, biosensing, and molecular computation amongst others. To fully realize the synergistic benefits from such organic–inorganic composites, especially when they constitute complex, multidimensional Forster resonance energy transfer (FRET) networks, a detailed understanding of the mechanisms that govern the individual components is imperative. Here, we demonstrate hybrid FRET systems comprising an initial QD scaffold/donor displaying DNA dendrimers decorated with dyes and which are capable of efficiently capturing UV light and transporting it to spectrally and spatially distant fluorophores via multistep FRET. We evaluate two primary strategies to conjugate the DNA-dendrimers to the QDs, namely covalent attachment of DNA to the termini of the QDs surface ligands and polyhistidine-based metal affinity coordination of modified DNA to the QDs ZnS shell surface. Analysis of the resulting FRET data shows that the dendritic arrangement of the dyes and the ability to place multiple dendrimer copies around the QDs nontrivial surface provides for significant energy transfer efficiencies of 20–25% through these multi-FRET step systems. In analyzing the properties of the conjugates, we further find that each assembly chemistry brings with it a series of benefits and liabilities that serve as mutual trade-offs and potential rules of thumb for designing future nanodevices based on these materials.

Restriction Enzymes as a Target for DNA-Based Sensing and Structural Rearrangement

DNA nanostructures have been shown viable for the creation of complex logic-enabled sensing motifs. To date, most of these types of devices have been limited to the interaction with strictly DNA-type inputs. Restriction endonuclease represents a class of enzyme with endogenous specificity to DNA, and we hypothesize that these can be integrated with a DNA structure for use as inputs to trigger structural transformation and structural rearrangement. In this work, we reconfigured a three-arm DNA switch, which utilizes a cyclic Förster resonance energy transfer interaction between three dyes to produce complex output for the detection of three separate input regions to respond to restriction endonucleases, and investigated the efficacy of the enzyme targets. We demonstrate the ability to use three enzymes in one switch with no nonspecific interaction between cleavage sites. Further, we show that the enzymatic digestion can be harnessed to expose an active toehold into the DNA structure, allowing for single-pot addition of a small oligo in solution.

Expanding molecular logic capabilities in DNA-scaffolded multiFRET triads

Dynamic rearrangement of DNA nanostructures provides a straightforward yet powerful mechanism for sequence-specific sensing and potential signaling of such interactions. These rearrangements are often interpreted in the context of Boolean logic gates as a means of both reflecting the underlying sensing and providing preliminary processing of the raw data. Here, we expand on previous work to optimize both the sensing and signal transduction of an initial DNA-triad sensor prototype. The core structure of this DNA triad consists of dye-labeled arms connected by 1, 2, or 3 single-stranded DNA linkers, whose presence and length alter the efficiency of Forster resonance energy transfer (FRET) between the dyes. The latter forms the basis for sensing through the use of DNA hybridization and displacement which result in structural rearrangements with each configuration correlated to a different logic state. Three different avenues were pursued to optimize the sensor function: (1) restructuring the connecting linkers and dye-choices in the original structure; (2) changing the mechanism of distance modulation between the arms; and (3) moving the signaling dyes to within the single-stranded portion of the structure. The first approach provided for improvements in FRET properties and the ability to reconfigure and switch the sensors between different types of Boolean logic gates such as going from INHIBIT 1 to Enabled OR by changing dyes, for example. The last approach proved to be the most versatile providing for the largest changes in FRET along with the ability to be repeatedly toggled and reset for multiple sequential sensing events. Switching could be completed in an isothermal manner with a near stoichiometric concentration of inputs and input complements. The continued development and potential applications of these and similar types of DNA sensors are discussed.

Reconfigurable DNA nanostructures for detection of multiple DNA and enzymatic inputs

The progress within the field of DNA nanotechnology has shown DNA to be an ideal material for the self-assembly of complex two-dimensional and three-dimensional structures. In addition to forming a wide range of structural geometries, DNA has been demonstrated as an exemplary scaffold. In this work we utilize this scaffolding ability to create photonic DNA switches that respond to both DNA and enzymatic inputs and produce complex logic based outputs.

Using DNA nanostructures to harvest light and create energy transfer and harvesting systems

DNA is a biocompatible scaffold that allows for the design of a variety of nanostructures, from straightforward double stranded DNA to more complex DNA origami and 3-D structures. By modifying the structures, with dyes, nanoparticles, or enzymes, they can be used to create light harvesting and energy transfer systems. We have focused on using Förster resonance energy transfer (FRET) between organic fluorophores separated with nanometer precision based on the DNAs defined positioning. Using FRET theory we can control the direction of the energy flow and optimize the design parameters to increase the systems efficiency. The design parameters include fluorophore selection, separation, number, and orientation among others. Additionally the use of bioluminescence resonance energy transfer (BRET) allowed the use of chemical energy, as opposed to photonic, to activate the systems. Here we discuss a variety of systems, such as the longest reported DNA-based molecular photonic wires (> 30 nm), dendrimeric light harvesting systems, and semiconductor nanocrystals integrated systems where they act as both scaffold and antennae for the original excitation. Using a variety of techniques, a comparison of different types of structures as well as heterogeneous vs. homogenous FRET was realized.

Utilizing homogenous FRET to extend molecular photonic wires beyond 30 nm (Presentation Recording)

Molecular photonic wires (MPWs) present interesting applications in energy harvesting, artificial photosynthesis, and nano-circuitry. MPWs allow the directed movement of energy at the nanoscopic level. Extending the length of the energy transfer with a minimal loss in efficiency would overcome an important hurdle in allowing MPWs to reach their potential. We investigated Homogenous Förster Resonance Energy Transfer (HomoFRET) as a means to achieve this goal. We designed a simple, self-assembled DNA nanostructure with specifically placed dyes (Alexa488-Cy3-Cy3.5-Alexa647-Cy5.5) at a distance of 3.4 nm, a separation at which energy transfer should theoretically be very high. The input of the wire was at 466 nm with an output up to 697 nm. Different structures were studied where the Cy3.5 section of the MPW was extended from one to six repeats. We found that though the efficiency cost is not null, HomoFRET can be extended up to six repeat dyes with only a 22% efficiency loss when compared to a single step system. The advantage is that these six repeats created a MPW which was 17 nm longer, almost 2.5 times the initial length. To confirm the existence of HomoFRET between the Cy3.5 repeats fluorescence lifetime and fluorescence lifetime anisotropy was measured. Under these conditions we are able to demonstrate the energy transfer over a distance of 30.4 nm, with an end-to-end efficiency of 2.0%, by utilizing a system with only five unique dyes.

Characterizing Functionalized DNA for Use in Nanomedicine

DNA as a structural nanomaterial demonstrates great potential as both an in vivo and in vitro designer platform for diagnostic and therapeutic medical use. Much of this work hinges on the ability of DNA to assemble into discrete, controlled structures that interact with, or bind to, other inorganic materials such as nanoparticles or biological molecules which include, for example, drugs and proteins. For these functionalized structures to be most effective, the spatial accuracy of their assembly must be precisely monitored and controlled. Clearly, to design and implement all forms of these functionalized DNA structures, a full characterization will ultimately be a critical necessity. With the current array of characterization techniques available, it can be difficult to choose one specific method especially considering that the efficacy can depend on the type of structure and the final application and environment in which the structure will be used. A review of current methods used for the characterization of complex DNA nanostructures can provide us with a greater understanding of which structures and applications will benefit from specific techniques. More importantly, it can also yield an understanding of which characterization methods can be used in concert to provide a more in depth and integrated understanding of a particular construct as a whole. Comparative characterization may also provide information on the many subtleties and nuances that are to be expected in these complex systems. In this critical overview of available characterization methods, we examine the techniques currently in use for these purposes.