The Wong Lab

Single-molecule Force Studies

 
 

relevent group publications

M.A. Koussa*, K. Halvorsen*, A. Ward, W.P. Wong, DNA Nanoswitches: A quantitative platform for gel-based biomolecular interaction analysis, Nature Methods (2015). (*equal contribution) [link] [pdf] [protocol] [videos] [author file]

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.



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).

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

Artwork: Mark Daws