A Window on the Nanoscale
Using DNA Origami to Contextualize Direct Observations of Enzymes in Action
- Fig. 1: Formation of DNA origami nanostructures.
- Fig. 2: RecA homology searching dynamics in DNA origami nanostructure. Direct measurement of interaction geometry (red bars) is in excellent agreement with MD simulations (blue line) [4,5].
- Fig. 3: RecA binds to ssDNA region (white arrow). UV photocleavable linker (yellow star) is used to initiate the reaction. The region of sequence homology is shown in green.
Microscopy typically comes in one of two flavors, providing either structural detail or functional context. Rarely does a microscopy technique bridge the void of form-to-function as well as high-speed-AFM. Augmenting this with nanoscale structures woven from DNA to host and control biological molecules, enables a variety of new explorations, including the direct characterization of rate, orientation, sequence specificity and interaction modes of individual enzymes, one at a time.
The Potential of High-speed AFM to Study Biological Interactions
Microscopy approaches to studying biological molecules can be broadly divided into two groups – those which provide high spatial resolution and thus aimed at understanding structural aspects, and those which provide high temporal resolution with the aim to understand functional aspects. For example, modern cryo-electron microscopy provides unparalleled spatial resolution able to reveal intricate structural details of fixed proteins. In contrast, state-of-the-art optical microscopes can operate with time-resolutions suitable to investigate the dynamics of biological processes and therefore provide insight on their functional context, yet these techniques are generally unable to resolve structural features at the molecular scale. Moreover, the majority of these approaches interrogate ensemble states and thus can only provide population-averaged information rather than single molecule detail.
In contrast high-speed atomic force microscopy (HS-AFM) offers both, the spatial and temporal resolutions to provide dynamic information at the single molecule level, thereby providing concurrent structural and function information yielding new insight. Furthermore, HS-AFM can operate in physiological environments and without fixation or labelling. However, it suffers a major caveat – the imposition of a solid-liquid interface. To limit the impact and interference of this interface, many sample preparation approaches have been developed, tackling the dichotomy of tethering the biological molecules of interest to the interface whilst leaving them free thus enough to interact with other molecules .
While the omission of labels is desirable to preserve physiological relevance, it restricts the AFM’s ability to derive species-specific information or coordinate the biological molecules in space without the aid of additional fiducials. To address this, we have introduced nanoscale objects woven from DNA molecules that can be used to host and control biological interactions at the single molecule level to augment the advantages of HS-AFM.
Nanoscale Experimental Platforms from DNA
In nature, DNA exists as the carrier of genetic information within many organisms. Information is faithfully encoded in the specific interactions between the nucleic acid subunits (bases) of the two strands of a DNA helix. The field of DNA nanotechnology uses DNA differently, simply as a structural polymer, harnessing the high-fidelity subunit-parings to specify unique interactions between collections of DNA molecules to form a designed shape (fig. 1). A robust approach to this is to use single stranded viral DNA as a scaffold which can be arbitrarily folded to fill the volume of a desired shape. A raster pattern is used to traverse the object, forming rows of parallel potential helices. Since the scaffold sequence is known, a set of short DNA strands can be synthesized such that they are complementary in part to two adjacent but non-canonical regions of the scaffold, thus “stapling” parallel helices together and cumulatively tying the scaffold into a rigid structure. This approach is termed DNA origami in likeness of the Japanese art of sequentially folded paper . Any arbitrary 2D, solid 3D, and wireframe architecture can be designed from different arrangements of the same DNA sequence, limited only by the length of the scaffold. As such, DNA origami is an incredibly versatile and robust method for constructing self-assembled nanoscale tools with which to augment our HS-AFM experiments.
For this HS-AFM application, DNA origami are formed into window-like structures within which DNA molecules can be hosted as reaction substrates for a variety of enzymes. The large surface-area of the DNA origami provides multiple attachment points to the imaging surface while leaving the reaction molecules within the frame free, thus satisfying the AFM sample preparation dichotomy. The DNA structure confines the enzymatic interaction to a predictable window readily discernable, and furthermore provides the required fiducial markers to characterize directly the reaction in situ. Moreover, additional DNA sequences or functional nucleic acid chemistry (such as photocleavable linkers) can be incorporated to augment further the experiments with in-built controls, the release of functional entities, or the creation of intermediate states via external means throughout the HS-AFM observation.
A Window on DNA Recombination
Here we focus on homologous recombination by the enzyme Recombinase A (RecA) – which is critical to genetic maintenance – to demonstrate the power of the DNA nanostructure-based HS-AFM approach. Despite decades of study, several functional aspects of RecA remain widely debated. RecA’s active state, where the protein is polymerized around single-stranded DNA (ssDNA), locates sequence homology between the encapsulated ssDNA and a double-stranded DNA (dsDNA) via a yet unknown mechanism.
For studying this process with HS-AFM, the DNA origami provides critical geometrical context to RecA complexes undertaking a homology search when compared to our previous observations, which highlighted larger-scale cooperativity but provided limited interpretation of the DNA–RecA interaction due to the random, unknown arrangements of the complexes on the surface . The predictable trajectory of the DNA substrate placed within the known dimensions of the DNA origami structure enabled the position of the RecA complex to be determined along the dsDNA at every time point. From this, the search rates and the specific mode of interaction were characterized on a single molecule level, highlighting the occurrence of facilitated diffusion along the dsDNA around regions of partial sequence homology to check for registration (fig. 2) . Furthermore, the interaction angle between the dsDNA and the RecA complex was quantified and shown to correlate well with that predicted by the MD docking simulations of others , indicating alignment of the dsDNA within the secondary binding site on the exterior of the complex .
To examine the subsequent recombination step in the RecA interaction, the DNA origami was adapted with a third, partially single-stranded, DNA molecule to act as the RecA polymer substrate. This was anchored in place with a UV photocleavable linker between the target homologous and the heterologous control dsDNA (fig. 3). Once RecA is formed on the ssDNA, the reaction is initiated by UV exposure and the complex is able to recombine the DNA molecules to form a triple-stranded joint, all of which can now be observed in real time by HS-AFM. This normally short-lived intermediate is frustrated as a non-hydrolysable ATP cofactor was used for these experiments. Not only can the final homologous product be observed, but the positions of the stable searching intermediates can be mapped out with reference to the DNA origami dimensions. Interestingly, these positions correlate well with the occurrence of sequence micro-homology within the dsDNA. As such, the use of DNA origami for these experiments enables us to map directly the sequence specific interactions of a single enzyme in a dynamic measurement.
A New Frontier for Single Molecule Studies
The advances of HS-AFM in tandem with the development of DNA origami-based approaches for hosting complex biological systems have opened up new avenues for investigating both form and function of biological molecules concurrently, thereby bridging the gap in understanding between the two. Furthermore, these are true single molecule investigations and thus offers the potential to study the variation in function between populations of an enzyme whose idioms would otherwise be averaged out. Importantly, the intrinsic versatility of the DNA origami provides a formidable route to expand the system beyond the study of individual enzymes to complex multi-component systems.
Andrew J. Lee1, Masayuki Endo2, Jamie K. Hobbs3, Christoph Wälti1
1Bioelectronics, The Pollard Institute, School of Electronic & Electrical Engineering, University of Leeds, UK
2Institute for Integrated Cell-Material Sciences, Kyoto University, Japan
3Department of Physics and Astronomy, University of Sheffield, UK
Dr. Andrew J. Lee
The Pollard Institute
School of Electronic & Electrical Engineering
University of Leeds
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 Andrew J. Lee, Rajan Sharma, Jamie K. Hobbs and Christoph Wälti: Cooperative RecA clustering: the key to efficient homology searcing, Nucleic Acids Research, 45: 11743–11751 (2017) doi: 10.1093/nar/gkx769
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 Andrew J. Lee, Masayuki Endo, Jamie K. Hobbs and Christoph Wälti: Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search, ACS Nano, 12: 272–278 (2018) doi: 10.1021/acsnano.7b06208
 Darren Yang, Benjamin Boyer, Chantal Prévost, Claudia Danilowicz and Mara Prentiss: Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure, Nucleic Acids Research, 43: 10251-10263 (2015) doi: 10.1093/nar/gkv883