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Using DNA Nanoswitches to Study Molecular Forces

New inexpensive technology platform enables multiplexed single-molecule analysis under force

The benchtop centrifuge force microscope device, consisting of the centrifuge force microscope unit (top), parts for transmitting the camera signal and a battery (right), fits into two standard buckets of a common laboratory centrifuge that are balanced by counterweights in the respective opposite buckets. Image: Wyss Institute

From the tension of contracting muscle fibers to hydrodynamic stresses within flowing blood, molecules within our bodies are subject to a wide variety of mechanical forces that directly influence their form and function.

Analyzing the responses of single molecules under conditions where they experience such forces could lead to a better understanding of many biological processes and potentially the development of more accurately acting drugs.

Until now, the experimental analysis of single-molecule interactions under force has been expensive, tedious and difficult to perform. It requires the use of sophisticated equipment, such as an atomic force microscope or optical tweezers, which permit analysis of only one molecule at a time.

Now, a research team led by Wesley Wong of Harvard’s Wyss Institute, Harvard Medical School and Boston Children’s Hospital has made a major advance. The scientists have developed an inexpensive method that allows analysis of the force responses of thousands of similar molecules simultaneously.

They report in Nature Communications how programmable DNA nanoswitches can be used in combination with a newly designed miniaturized centrifuge force microscope as a highly reliable tool to observe thousands of individual molecules and their responses to mechanical forces in parallel.

“This new combined approach will allow us and others to examine how single-molecule complexes behave when they are thrown out of their equilibrium by the tunable force generated in our newly designed centrifuge force microscope,” said Wong, who is the study’s senior author.  

Wong is an associate faculty member at the Wyss Institute for Biologically Inspired Engineering and an HMS assistant professor of biological chemistry and molecular pharmacology at Boston Children’s. “By basing this instrument on something that most researchers already have and use—the benchtop centrifuge—we hope to make single-molecule force measurements accessible to almost everyone.”

The picture on the top shows a DNA nanoswitch that forms a looped structure when a bond is formed between the attached reactive components (receptor-ligand pair shown in red and green); at one end it is attached to the sample stage and at the other to a bead (top). By applying centrifugal forces to the bead in the CFM device, the bond between the reactive components can be repeatedly ruptured, opening up the loop and increasing the length of the DNA tether (bottom), enabling highly reliable measurements of molecular interactions. In the centrifuge force microscope, many beads can be interrogated in parallel, enabling high-throughput single-molecule measurements (bottom left). In the video in the bottom right, the camera captures these rupture events in real time by registering the bead at a different spot. Image: Wyss Institute

Earlier efforts led by Wong at the Rowland Institute at Harvard introduced the first centrifuge force microscope in 2010, which was a highly specialized instrument that carried out high-throughput precision force measurements on single molecules by tethering them to beads and pulling at them using centrifugal force. In this latest iteration of the centrifuge force microscope, Wong and his team developed a way to carry out the same technique with similar precision, using a small, inexpensive microscope made from easy-to-assemble elements and 3D-printed parts that can be inserted into the swinging bucket of a standard benchtop centrifuge found in virtually all biomedical research laboratories.

In addition, the team increased the robustness and accuracy of the assay by integrating thousands of so-called DNA nanoswitches, linear DNA strands with pairs of interacting molecules that are associated with two sequences in their middle. By binding to each other, the nanoswitches create an internal DNA loop; their ends are tethered to the surface of the sample on one side and to beads on the other.

“By applying a defined range of centrifugal forces to the beads, we can provoke the rupture of the molecular complexes generating the looped DNA structures which will be registered by the camera-coupled lens. Importantly, using DNA nanoswitches as a stable scaffold allows us to repeat this process multiple times with the very same molecule in temperature-controlled conditions, which greatly enhances our accuracy in determining the heterogeneity that a single molecular interaction can display,” said Darren Yang, the first author of the study and a graduate student on Wong’s team.

In future research, bead-associated DNA nanoswitches can be employed to repeatedly assemble and rupture many different biomolecular complexes and to define the mechanical forces that control them. “The integrated DNA nanoswitches are very modular, and can be functionalized with many different biomolecules in essentially a plug-and-play fashion, to enable a wide variety of molecular interactions to be studied with high throughput and reliability,” Wong said.

Next, the Wyss scientists are planning to apply their DNA nanoswitch-enhanced miniature centrifuge force microscope to investigate select biomedically relevant and force-dependent molecular interactions such as protein interactions governing blood clotting or hearing.

“Wong’s team has created a new technology platform that greatly reduces the cost of single-molecule force analysis and makes it widely accessible to the scientific community. In addition to increasing our understanding of basic molecular structure-function relations, it may prove to be a valuable tool for drug development,” said Wyss Institute Founding Director Donald Ingber, the Judah Folkman Professor of Vascular Biology at HMS and the vascular biology program at Boston Children’s Hospital. 

Adapted from a Wyss Institute news release.