New computational framework for crisscross fabrication of micrometer-scale DNA megastructures with nanoscale precision broadens accessibility to this powerful nanotechnology
By Benjamin Boettner
(BOSTON) — 44 years ago, Nadrian Seeman published his groundbreaking ideas on using DNA as a structural material, expanding DNA’s significance way beyond that as a carrier of genetic information. Since then, the steadily growing field of DNA nanotechnology has seen numerous innovations. One of them was the DNA origami technique, which enables researchers to fold a single long strand of DNA into a desired 2D or 3D shape.
The group of William Shih, Ph.D., at the Wyss Institute at Harvard University and Dana-Farber Cancer Institute (DFCI) was the driving force in expanding the initial 2D DNA origami concept into 3D geometries. Their new self-assembly methods allowed them to build 3D multi-layered DNA structures that are more stable, rigid, and resilient against harsh chemical and biological environments. DNA nanotechnologists quickly envisioned future devices like new vehicles for targeted drug delivery, autonomously functioning nano-robotic machines, and ultra-precise electronic devices. A recent example, the Wyss Institute’s DoriVac project, led by Shih, has used DNA origami to precisely organize vaccine components at the nanoscale to enhance immune activation against cancer.
However, Shih and other scientists hypothesized that pushing the envelope on the size and 3D complexity of DNA structures even further, while preserving the unique capabilities of DNA origami, could lead to DNA megastructures that could be applied to even more complex tasks. “The possibility of designing DNA megastructures on the scale of micrometers with features that can be arranged with nanoscale precision could potentially open up paths for sophisticated applications. These include optical devices that can modulate light at visible wavelengths, cell-DNA interfaces that function as “cell mimics” to program, for example, the behavior of certain immune cells in disease, or molds and scaffolds for assembling tissues with programmable shapes and functions,” said Shih, who is a Founding Core Faculty member at the Wyss Institute and Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and DFCI.
The possibility of designing DNA megastructures on the scale of micrometers with features that can be arranged with nanoscale precision could potentially open up paths for sophisticated applications. These include optical devices that can modulate light at visible wavelengths, cell-DNA interfaces that function as ‘cell mimics’ to program, for example, the behavior of certain immune cells in disease, or molds and scaffolds for assembling tissues with programmable shapes and functions.
In fact, in 2021, his group pioneered a new nanofabrication concept they called “crisscross polymerization” and applied it to weave together DNA nanoribbons from “slats” made of elongated strands of DNA. Starting from a tiny seed, the ribbons can keep growing until they become several micrometers in length. This new fabrication principle has the potential to be leveraged in diagnostic assays in which the presence of rare biomarkers of disease in patient samples could kickstart a super-fast crisscross reaction resulting in the assembly of an easily detectable DNA nanoribbon as a diagnostic read-out. Taking the concept a step further, in 2023 the team applied the principles of crisscross polymerization to build significantly larger and more complex structures, this time using slats that are made from arrays of entire, interlinked DNA origami structures. The possible complexity of these “crisscross DNA megastructures” moved the envisioned light, cell, and tissue programming technologies closer to reality. However, “there is a difference between demonstrating that something can be done in principle, and enabling it to be done with high efficiency, with very low error rates, and in many more research labs,” said Shih. “The challenge of avoiding unwanted DNA binding events in self-assembling DNA nanostructures just becomes magnified enormously in the fabrication of crisscross DNA megastructures.”
Getting a handle on crisscross DNA megastructures
Now, his team, spearheaded by Research Fellows Matthew Aquilina, Ph.D., and Florian Katzmeier, Ph.D., published in Nature Communications a computational framework that standardizes the design and fabrication of crisscross megastructures to make the nanotechnology more broadly accessible. During crisscross polymerization, multiple origami slats are first assembled into a grid on the seed structure. Then, a second antiparallel grid forms at a 90° angle. This step repeats itself, over and over again, in crisscross fashion until the desired megastructure reaches its final size and shape. Key to megastructure assembly is that all slat interactions are enabled by short so-called “handle” DNA sequences that function as molecular Velcro to connect specific positions on origami slats in one grid layer to corresponding positions on slats in the next grid layer.
The precision with which the overall megastructure assembly occurs thus hinges on the accuracy with which all handles function; handles that bind to false counterparts in the structure disrupt the self-assembly process, reducing the overall yield of correctly finished crisscross megastructures.” In our recent study, we minimized such handle mismatches by developing an evolutionary algorithm that optimizes handle selection and origami design from the get-go. Embedded in a user-friendly graphical interface that provides users with control over the entire design and fabrication process, this is an important step toward putting this DNA nanotechnology in the hands of many more researchers and democratizing crisscross DNA megastructure fabrication and use,” said Shih.
After users have laid out their desired crisscross megastructure using their custom software package, #-CAD, the algorithm tests multiple candidate handle sequences from an existing library at each node that connects slats from different grid layers. It penalizes those that would lead to mismatches, takes the best ones, makes slight alterations to them by swapping in and out different handles (mutation), and iterates this operation through multiple rounds until it has found an optimum. “The algorithm evolves all the thousands of handles in a highly complex crisscross megastructure in parallel,” said Aquilina. “By predicting a handle set that has the highest probability of assembling a well-formed megastructure, it removes a central bottleneck in an otherwise daunting design process, basically solving a multimillion-piece puzzle in 3D,” said Aquilina.
1/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
2/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
3/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
4/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
5/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
6/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
7/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
8/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
9/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University 
10/10 The team used their easily accessible computational framework, #-CAD, to design and fabricate a series of complex Crisscross DNA megastructures from start to finish. Shown here is a gallery that pairs various #-CAD-assisted design graphics with microscopic images of the final structures. The hexagonal megastructures on the right include additional fluorescent elements that demonstrate how megastructures could be functionalized. Credit: Wyss Institute at Harvard University
Fabrication automated, results on view
But the advance of the team’s study doesn’t stop here. #-CAD also provides fabrication-ready outputs, including instructions for robotic handlers that take on the tedious pipetting work for retrieving selected handle DNA from multi-well plates and combining them with the other DNA components. “This allows the fabrication of crisscross megastructures to be largely automated, somewhat similar to 3D printing. Our goal was to hide as much of the underlying complexity as possible without taking control away from the researcher. You should be able to think about the design you want to build rather than spending your time manually keeping track of thousands of DNA sequences and pipetting steps,” said Katzmeier. “The combined capabilities of our open-source #-CAD computational platform massively streamline the workflow and reduce the costs and time needed for prototyping such megastructures for diverse future applications.” Demonstrating the power of #-CAD, the team fabricated a gallery of megastructures with vastly different geometries and molecular cargo that serve as technical proof and may further stimulate the creative energy of DNA nanotechnologists.
This allows the fabrication of crisscross megastructures to be largely automated, somewhat similar to 3D printing. Our goal was to hide as much of the underlying complexity as possible without taking control away from the researcher. You should be able to think about the design you want to build rather than spending your time manually keeping track of thousands of DNA sequences and pipetting steps.
“This recent breakthrough by William Shih’s team will not only allow other scientists to begin to explore the potential use of DNA megastructures as materials for building, control, and molecular detection, but also provide a way to integrate automation and eventually AI into the process. In the spirit of the Wyss’ Molecular Robotics Initiative, they thus broke down an intimidating barrier to bringing this exciting technology to full fruition,” said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.
Other authors on the study are Minke Nijenhuis, Siyuan Stella Wang, Corey Becker, Yichen Zhao, Su Hyun Seok, Julie Finkel, Huangchen Cui, Jaewon Lee, and Seungwoo Lee. The study was funded by the Wyss Institute Northpond Alliance Director’s Fund and Wyss Institute Molecular Robotics Initiative, DFCI Claudia Adams Barr Program for Cancer Research, U.S. Department of Energy (award DE-SC0024136), Sloan Foundation (grant ID G-2021-16495), German Research Foundation’s Walter Benjamin Program (project 553862611), Korea-US Collaborative Research Fund (grant RS-2024-00468463), Novo Nordisk Foundation (grant NNF23OC0084494), and Carlsberg Foundation (grant CF23-1125), as well as a UK Medical Research Council Precision Medicine Transition Fellowship (MR/N013166/1).