We’ve developed a nanoscale experimental platform that enables kinetic and equilibrium measurements of a wide range of molecular interactions using a gel electrophoresis readout. Programmable, self-assembled DNA nanoswitches serve both as templates for positioning molecules, and as sensitive, quantitative reporters of molecular association and dissociation.
DNA NANOSWITCHES: A QUANTITATIVE PLATFORM FOR SINGLE-MOLECULE AND GEL-BASED BIOMOLECULAR INTERACTION ANALYSIS
Whether one is designing new drugs to target cancer cells, or trying to understand the basic mechanisms underlying disease, a central question that always arises is: How do these biological components interact?
While a number of sophisticated methods have been developed to answer this question, issues of cost, throughput, and complexity can serve as barriers to their widespread use. We’ve solved these challenges by developing a nanoscale tool that can report molecular interactions by simply changing its shape, enabling interaction measurements to be made by anyone, using only common and inexpensive laboratory reagents. By harnessing the inherent properties of DNA, we have been able to build these nanoscale switches using self-assembly, making this technology both affordable, and easy to use.
By affixing molecules of interest to a linear scaffold of DNA, interaction events between these molecules are converted into a change in shape. For example the formation of a bond between two molecules will cause the scaffold to switch from a linear, or open, state, into a looped, or closed, state. The states of the nanoswitch can be read out using one of the most standard, and inexpensive laboratory techniques, gel electrophoresis. They can also be read out using single-molecule methods. By watching how the nanoswitches transition from open to closed, or closed to open over time, we characterize not only the strength of the interaction, but also its kinetics properties.
In its current form, this approach enables researchers to make accurate biochemical measurements in a simple and cost effective way, which should accelerate research discoveries in a broad range of fields. Furthermore, this approach is incredibly versatile, as we have demonstrated by studying a variety of different molecular systems, ranging from interactions between single pairs of atoms, to complex 4 body interactions. Further extensions of this method will also enable sophisticated screening methods for the discovery of new and better-targeted therapeutics.
It is our hope that this powerful, yet simple and inexpensive approach, will enable everyone to make new research discoveries in their labs. To help others get started with this method, we’ve developed a step-by-step tutorial that walks you through the its use to study both the kinetic and thermodynamic properties of a biomolecular system--check out the wyss.harvard.edu/nanoswitch below for detailed video protocols.
Gel electrophoresis, a common laboratory process, sorts DNA or other small proteins by size and shape using electrical currents to move molecules through small pores in gel. The process can be combined with novel DNA nanoswitches, developed by Wyss Associate Faculty member Wesley Wong, to allow for the simple and inexpensive investigation of life’s most powerful molecular interactions. Credit: Wyss Institute at Harvard University
Kinetic measurements using DNA nanoswitches: a The two states (bound and unbound) of the DNA nanoswitches can be distinguished by gel electrophoresis. b With two biotins integrated into the nanoswitch, loop formation begins when unlabeled streptavidin is introduced and progresses over time as evidenced by increasing intensity in the bound (looped) band across different lanes of a gel (bottom). The growth curve is fit with a kinetic model to determine the on-rate. c Addition of excess biotin blocks loop formation, making bond rupture irreversible, which leads to the exponential decay of nanoswitches from the bound state to the unbound state, as shown by the decreasing intensity in the unbound band across different lanes of a gel (bottom). d Temperature dependence of on-rates and off-rates for the biotin-streptavidin interaction at 150 mM NaCl.
Multistate kinetic analysis: a A nanoswitch functionalized with two digoxigenin molecules and one biotin molecule can adopt 5 discernable states upon addition of a bispecific receptor. All 5 topological states, A-E, can be resolved within a single lane of an agarose gel. These bands can be fit globally with a single fit of a sum of skewed Gaussian curves. The black curve represents the median pixel intensity, the dashed red curve represents the fit which is the sum of 5 skewed Gaussians, and the individual skewed Gaussians are shaded by state. b A reaction diagram illustrating the possible transitions between each of the 5 states. c (left) on-rate measurements indicating the value of each state at 20 different time points. Solid curves indicate the result of a global fit of all states to the kinetic model illustrated in c. (right) off-rate measurements indicating the value of each state at 12 different time points. Solid curves indicate the result of a global fit of all states to the kinetic model illustrated in b. These fits taken together allowed for the determination of all rate constants from 32 lanes which can be run on a single gel
RELEVANT GROUP PUBLICATIONS
Single-molecule mechanical fingerprinting with DNA Nanoswitch Calipers
P Shrestha*, D Yang*, TE Tomov, JI MacDonald, A Ward, HT Bergal, E Krieg, S Cabi, Y Luo, B Nathwani, A Johnson-Buck, WM Shih^, WP Wong^. Nat. Nanotechnol. (accepted). PDF
Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have significantly impacted clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Toward this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA Nanoswitch Caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution.... Read more ›
Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection
Basic research and medical diagnostics rely on the ability to detect and quantify specific proteins in biological fluids. While numerous current detection techniques exist, these are often limited by trade-offs between ease of use, sensitivity, and cost. Here, we present the nanoswitch-linked immunosorbent assay (NLISA), an accessible, sensitive, and low-cost detection platform that is based upon nanoscale devices that change confirmation upon binding a target protein. NLISA is surface-free and includes a kinetic-proofreading purification step, enabling both enhanced sensitivity and the ability to accurately distinguish between similar proteins from different strains of the same virus or that differ by only a single mutation. Our method is also readily transferable to point-of-care devices due to an easy readout and few hands-on steps. Read more ›
DNA Nanoswitches: A quantitative platform for gel-based biomolecular interaction analysis
MA Koussa*, K Halvorsen*, A Ward, WP Wong. Nat. Methods 12, 123-126 (2015). PDF, Protocol, Videos
We introduce a nanoscale experimental platform that enables kinetic and equilibrium measurements of a wide range of molecular interactions using a gel electrophoresis readout. Programmable, self-assembled DNA nanoswitches serve both as templates for positioning molecules and as sensitive, quantitative reporters of molecular association and dissociation. We demonstrated this low-cost, versatile, 'lab-on-a-molecule' system by characterizing ten different interactions, including a complex four-body interaction with five discernible states. Read more ›