Molecular Biosensors
DNA nanoswitches for detecting biomarkers
The development and application of DNA nanoswitches for biosensing has become a major focus of the lab. The detection of biologically relevant markers is important toward the diagnosis of diseases, eventually leading to their treatment. Biomarkers such as small noncoding RNAs and proteins, for example, are expressed in higher (or lower) levels in diseased states. If a biosensor can detect these changes in levels, we may be able to detect the onset of a particular disease in its early stages.
The evolution of this technology has been somewhat serendipitous. The DNA nanoswitches were originally developed by Ken Halvorsen and Wesley Wong as a sort of molecular lanyard to attach two molecules together for single molecule experiments. At one point during the development, after many failed attempts at constructing such a lanyard by various means, we were discussing the hot new topic of DNA origami. Right on the cover of Nature, in bold letters staring at us was “Nanoscale shapes the easy way”. It was tempting enough that we took the plunge and tried it out. As the field was moving from 2D to 3D nanoscale objects, we joked that if successful we’d be moving the field backward with our 1D origami. We succeeded in that and made our binary DNA switch, a simple linear DNA duplex in the “off”state and a duplex with a loop in the “on”state.
At that point we noticed that running our DNA nanoswitches on a gel produced two separate bands for the on and off states. This one simple observation surprised us (don’t laugh, we were engineers and physicists – molecular biology newbies), and led us in this completely different direction. We recognized then that these relatively low cost DNA nanoswitches could be used to detect molecules and analyze molecular interactions using nothing more than gel electrophoresis. Our first paper demonstrating some of these capabilities came out in 2015 in Nature Methods.
Since that time, our lab has focused on detection and analysis of RNAs. We have recently validated this system for the detection of microRNAs with an assay we nicknamed miRacles (microRNA activated conditional looping of engineered switches). This assay provides detection of specific microRNA sequences from cellular RNA extracts and we showed that it could monitor upregulation of certain microRNAs in different stages of muscle cell growth. Collaboratively with the Wong lab we are expanding to develop a multiplexed assay that can detect a pancreatic cancer biomarker panel consisting of both proteins and microRNAs in a single assay.
We encourage other users interested in DNA nanoswitches to reach out to us. We hold an annual hands-on workshop in March, and we are happy to provide help and samples to get people/labs started.
DNA nanoswitches. Artistic representation of the on (looped) and off (linear) states of the nanoswitch. Image by Mark Daws.
Single Molecule Biophysics
The Centrifuge Force Microscope enables massively parallel single molecule experiments
Centrifugal force-based single molecule experiments. Artistic representation of the centrifuge force microscope. Image by Mark Daws.
The Centrifuge Force Microscope (CFM) was borne out of first-hand frustration with optical tweezers and the typical one-at-a-time data collection for force-based single molecule experiments. Wesley Wong and Ken Halvorsen (lab mates in grad school) co-conspired to develop an alternative that could allow multiplexing of single-molecule experiments. It started as a bit of a joke, why don't we just throw a microscope in a centrifuge and use the centrifugal force to perform thousands of single-molecule pulling experiments at once? Fate had it that Wesley became a junior fellow at The Rowland Institute at Harvard, where they encourage and support risky ideas. He brought Ken on as a postdoc and together they made the concept a reality.
Even among risk takers at the Rowland, most of their colleagues thought it was a crazy idea. Between them they secretly also admitted a high chance of failure - it was easy to think of dozens of technical problems that could derail the project. And indeed dozens of technical problems did come up but they all turned out to be solvable. The pair had too many failed designs and experiments to remember (luckily no injuries considering the kinetic energy), but eventually got to a working prototype and in 2010 published their work in Biophysical Journal, demonstrating thousands of individual single-molecule experiments carried out in parallel rather than one-at-a-time.
In the years since, we have come a long way from that prototype CFM, owing to efforts in Wesley's lab (now at Boston Children's Hospital/Harvard Med), Ken's lab at The RNA Institute, and more recently in third party labs that have started to adopt the technology. The instrument is now compatible with standard benchtop centrifuges and operable without an advanced degree and months/years of experience. We continue to make improvements to the CFM instrument, with focus on cost, ease of use, and implementing new features.
At the same time, we are working on a number of applications that benefit from the high-throughput of the CFM. Being at The RNA Institute and surrounded by RNA researchers, we lean toward RNA applications. Currently, our efforts include fundamental studies of RNA and DNA interactions, ribozyme interactions and cleavage, mechanical properties of DNA and RNA nanostructures, and single-molecule mechanical sensing.
If you'd like to learn more about the CFM please feel free to contact us. We have helped many labs get started building CFMs and are happy to continue doing so.
Functional DNA nanotechnology
Working toward biomedical applications of DNA nanostructures
In another area of focus, we use DNA to build things such as nanoscale objects and devices. DNA nanotechnology, as the field is called, deals with the design of synthetic DNA strands that can come together and assemble into tiny parts that are useful in different applications. In our lab, we work on both the structural and functional aspects of DNA nanotechnology.
In the structural focus, we look into various assembly parameters and DNA designs and the properties of these materials, such as biostability. On the functional side, we collaborate with chemists on the floor to add chemical groups on our DNA objects, that can be used to attach or release a cargo molecule, for example.
One key area of DNA nanotechnology is in drug delivery applications. In this area, we have worked on chemical strategies to place functional molecules at different locations. More recently, we have developed multi-functional DNA nanostructures that can also release payloads with biological and physical triggers (think RNA molecules and light). We have experimented with a few different DNA motifs, including a few of our own DNA/RNA hybrid structures. We also have a special interest in biological stability of DNA nanostructures, since drug delivery is unlikely to be successful if structures fall apart in biological fluids. We are working to understand how structural and chemical features of DNA nanostructures can act to protect structures. The long-term goal for all of this work is to develop functional DNA nanostructures that can solve real biomedical problems.
Paranemic crossover DNA. The four-stranded paranemic crossover (PX) DNA (foreground) exhibits remarkable resistance against nucleases (gold spheres) compared to regular double-stranded DNA that are quickly destroyed (background). Image by Alex Tokarev.