Collaborations between nanotechnologists, synthetic biologists, and computer scientists create nanoscale tools that could revolutionize fields from cancer diagnostics to materials science
By Lindsay Brownell
(BOSTON) — DNA has often been compared to an instruction book that contains the information needed for a living organism to function, its genes made up of sequences of the nucleotides A, G, C, and T echoing the way that words are composed of strings of letters. DNA, however, has several advantages over books as an information-carrying medium, one of which is especially profound: based on its nucleotide sequence alone, single-stranded DNA can self-assemble, or bind to a complementary DNA strand, to form a complete double-stranded helix, without human intervention. That would be like printing the instructions for making a book onto loose pieces of paper, putting them into a box with glue and cardboard, and watching them spontaneously come together to create a book with all the pages in the right order.
But just as paper can also be used to make cups, origami animals, and even the walls of houses, DNA is not limited to its traditional purpose as a set of genetic blueprints from which proteins are made – it can be engineered to self-assemble into different shapes that serve different functions, simply by controlling the order of As, Gs, Cs, and Ts along its length. A group of scientists at the Wyss Institute for Biologically Inspired Engineering at Harvard University is investigating this exciting property of DNA molecules, asking, “What types of systems and structures can we build with them?”
They’ve decided to build robots.
At first glance, there might not seem to be much similarity between a strand of DNA and, say, a Roomba™ or Rosie the Robot from The Jetsons. “Looking at DNA versus a modern-day robot is like comparing a piece of string to a tractor trailer,” says Wyss Faculty member Wesley Wong. However, despite the vast difference in their physical form, robots and DNA share the ability to be programmed to complete a specific function – robots with binary computer code, DNA molecules with their nucleotide sequences.
“We’re not used to thinking about molecules inside cells doing the same things that computers do. But they’re taking input from their environment and performing actions in response – a gene is either turned on or off, a protein channel is either open or closed, etc. – in ways that can resemble what computer-controlled systems do,” continues Wong.
Recognizing that commonality, the Wyss Institute created the cross-disciplinary Molecular Robotics Initiative in 2016, bringing together researchers with experience in the disparate disciplines of robotics, molecular biology, and nanotechnology to collaborate and help inform each other’s work to solve the fields’ similar challenges. The Initiative was founded by Wong and fellow Wyss Faculty members William Shih, Peng Yin, and Radhika Nagpal, and now includes other Wyss scientists and support staff.
“Molecules can do a lot of things on their own that robots usually have trouble with (moving autonomously, self-assembling, reacting to the environment, etc.), and they do it all without needing motors or an external power supply,” explains Wyss Founding Director Don Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as a Professor of Bioengineering at Harvard SEAS. “Programmable biological molecules like DNA have almost limitless potential for creating nanoscale devices and systems that could be transformational in a large number of industries and fields.”
Molecular Robotics capitalizes on the recent explosion of technologies that read, edit, and write DNA (like next-generation sequencing and CRISPR) to manipulate DNA and its single-stranded cousin, RNA, to create new nanoscale structures and devices that serve a variety of functions. “We essentially treat DNA not only as a genetic material, but as an incredible building block for making molecular sensors, structures, computers, and actuators, all of which self-assemble in a way that today’s traditional robots can’t,” says Tom Schaus, a Staff Scientist at the Wyss Institute who works on Molecular Robotics.
Many of the group’s early projects taking advantage of DNA-based self-assembly were static structures. These include DNA folded into 3D origami-like objects and DNA “bricks” whose nucleotide sequences allow their spontaneous assembly into a specified shape, like tiny Lego™ bricks that are pre-programmed to put themselves together to create a castle. The most recent iteration of DNA bricks can incorporate as many as 30,000 unique DNA strands in a single complete structure, and could enable the creation of novel devices for electronics, photonics, and nanoscale machines.
The reliable specificity of DNA and RNA’s nucleotide pairing (A always binds with T or U, C always with G) allows for not only the construction of nanoscale structures, but also the programming of dynamic systems that achieve a given goal. For example, Molecular Robotics scientists have created a novel, highly controllable mechanism that automatically builds new DNA sequences from a mixture of short fragments in vitro. It utilizes a set of DNA strands folded into a hairpin shape with a single-stranded “overhang” sequence dangling off one end of the hairpin. The overhang sequence can be programmed to bind to a complementary free-floating fragment of DNA (a “primer”) and then fall off, after extending the primer with a newly synthetized sequence that is identical to part of the hairpin sequence. This hairpin sequence can then serve as a new primer for another hairpin containing a different sequence, and the process can be repeated many times to create long DNA product strands through a technique called “Primer Exchange Reactions” (PER).
We’re trying to push the limits of these really dumb little molecules to get them to behave in sophisticated, collective ways – it’s a new frontier for DNA nanotechnology.
Not only can PER be used to synthesize DNA sequences automatically, it can be programmed such that it only occurs in the presence of signal molecules, such as specific RNA sequences, thus allowing the system to respond to the molecular cues in the environment much like today’s commercial robots respond to verbal and visual cues. The PER product strand can in turn be programmed to enzymatically cut and destroy particular RNA sequences, record the order in which certain biochemical events happen, or generate components for DNA structure assembly.
PER reactions can also be combined into a mechanism called “Autocycling Proximity Recording” (APR), which records the geometry of nano-scale structures in the language of DNA. In this technique, unique DNA hairpins are attached to different target molecules and, if any two targets are close enough together, a reaction between the two hairpins bound to them produces new pieces of DNA that contain a record of both hairpins’ sequences, allowing the shape of the underlying structure to be determined by sequencing that novel DNA.
Another tool, called “toehold switches,” can be used to exert complex and precise control over the machinery inside living cells. Here, a different, RNA-based hairpin is designed to “open” only when it binds to a specific RNA molecule, exposing a gene sequence in its interior that can be translated into a protein that then performs some function within the cell. These synthetic circuits can even be built with logic-based sequences that mimic the “AND,” “OR,” and “NOT” system upon which computer languages are based, allowing their opening to be tuned to highly complex biological environments. Such an approach could potentially induce cells that are deficient in a given protein to produce more of it, or serve as a synthetic immune system that, when it detects a given problem in the cell, produces a toxin that kills it to prevent it from spreading an infection or becoming cancerous.
The potential applications of such interaction with and control over biological are seemingly endless. In addition to the previously mentioned tools, Molecular Robotics researchers have created loops of DNA attached to microscopic beads to form “calipers” that can both measure the size, structure, and stiffness of other molecules, and form the basis of inexpensive protein recognition tests. Another recent advance is folding DNA origami from a single piece of DNA rather than from hundreds of shorter components, which allows large nanostructures to be produced from material generated in vivo.
Some of these many academic projects are already moving into the commercial sector. They include a low-cost alternative to super-resolution microscopy (DNA-PAINT) that has ultra-high resolution of up to 5 nanometers and achieves precise quantification of the number of molecules, as well as highly multiplexed imaging of many different targets in the same cell (Exchange-PAINT). The technology can also work with thick samples and even tissues.
One of the major benefits of engineering molecular machines is that they’re tiny, so it’s relatively easy to create a large amount of molecular devices that each perform the same task. Getting simple individuals to interact with each other to achieve a more complex, collective task (like relaying the information that cancer has been found), however, is a significant challenge, and one that the roboticists in Molecular Robotics are tackling at the macroscopic scale with inch-long “Kilobots.”
Taking cues from colonies of insects like ants and bees, Wyss researchers are developing swarms of robots that are themselves limited in function but can form complex shapes and complete tasks by communicating with each other via reflected infrared light. The insights gained from studies with the Kilobots are likely to be similar to those needed to solve similar problems when trying to coordinate molecular robots made of DNA.
“In swarm robotics, you have multiple robots that explore their environment on their own, talk to each other about what they find, and then come to a collective conclusion. We’re trying to replicate that with DNA but it’s challenging because, as simple as Kilobots are, they’re brilliant compared to DNA in terms of computational power,” says Justin Werfel, a research scientist in Molecular Robotics. “We’re trying to push the limits of these really dumb little molecules to get them to behave in sophisticated, collective ways – it’s a new frontier for DNA nanotechnology.”
Given the magnitude of the challenge and the short time the Molecular Robotics Initiative has existed, it is already making significant progress, with more than two dozen papers published and two companies founded around its insights and discoveries (Ultivue, for high-precision tissue imaging and NuProbe, for nucleic acid diagnostics). It may take years of creative thinking, risk taking, and swapping ideas across the members’ different expertise areas before a molecule of DNA is able to achieve the same task on the nanoscale that a robot can do on the human scale, but the team is determined to see it happen.
“Our vision with Molecular Robotics is to solve the big, complex problems humanity currently faces using programmable molecular tools,” says Shih, one of the founding directors for the Initiative. “It’s an idea that definitely goes against the current status quo, and we’re lucky enough to be pursuing it here at the Wyss Institute, which brings people together from different fields to pursue innovative ideas and create new things that wouldn’t exist otherwise.”
“We envision an exciting technological future where programmable molecular structures and devices, or molecular robots, will be engineered to carry out specified tasks at the molecular level,” adds Yin, another founding director. “These programmable systems will enable us to freely interact with the molecular world and achieve wide-ranging goals including sophisticated nanomanufacturing, efficient data storage and energy production, precise observation and manipulation of biological processes, and effective disease diagnostics and therapeutics”
1/4 Primer Exchange Reaction (PER) cascades enable the autonomous growth of single-stranded DNAs. On the top, a ‘catalytic PER hairpin’ binds a first ‘primer’ (shown as a short gray strand), triggers its elongation with a sequence encoded by the hairpin itself, and releases it to start another cycle with the extended primer, and so forth, until a long transcript is generated. Credit: Wyss Institute at Harvard University 
2/4 Illustration of an RNA-based ‘ribocomputing’ device that makes logic-based decisions in living cells. The long gate RNA (blue) detects the binding of an input RNA (red). The ribosome (purple) reads the gate RNA to produce an output protein. Credit: Alexander Green / Arizona State University 
3/4 A DNA nanoswitch forms a looped structure when a bond is formed between the attached receptor-ligand pair (shown in red and green); one end is attached to a solid base and the other to a bead (top). By applying centrifugal forces to the bead, the bond between the bound components can be repeatedly ruptured, opening up the loop and increasing the length of the DNA tether, enabling highly reliable measurements of molecular interactions. Many beads can be used in parallel (bottom left), enabling high-throughput single-molecule measurements, which cameras can capture in real time (bottom right). Credit: Wyss Institute at Harvard University 
4/4 The most recent advance by the Molecular Robotics Initiative is a method to construct DNA origami out of single-stranded rather than traditional double-stranded DNA. Credit: Wyss Institute at Harvard University
Click on the links below to explore research from the Molecular Robotics Initiative.
- Nanoscale origami from DNA
- A 100-fold leap to GigaDalton DNA nanotech
- Autonomously growing synthetic DNA strands
- High-fidelity recording of molecular geometry with DNA “nanoscopy”
- Programming cells with computer-like logic
- Democratizing high-throughput single molecule force analysis
- Single-stranded DNA and RNA origami go live
- Capturing ultrasharp images of multiple cell components at once
- A self-organizing thousand-robot swarm
- Discrete Molecular Imaging
The Molecular Robotics Initiative was founded by:
- William Shih, Ph.D., Professor of Biological Chemistry and Molecular Pharmacology BCMP at HMS and Dana-Farber Cancer Institute
- Peng Yin, Ph.D., Professor of Systems Biology at HMS
- Wesley Wong, Ph.D., Assistant Professor of (BCMP) at Harvard Medical School (HMS) and Investigator at Boston Children’s Hospital
- Radhika Nagpal, Ph.D., Fred Kavli Professor of Computer Science at Harvard’s John A. Paulson School of Engineering and Applied Sciences (SEAS)