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DNA nanostructures grow up to become micron-scale megastructures

New DNA nanofabrication approach enables high-yielding assembly of complex multi-origami megastructures, with potential for many biological and non-biological applications

By Benjamin Boettner

This animation explains how the newly invented crisscross origami method can be used to build functionalized micron-scale DNA megastructures composed of many unique DNA origami “slats,” each with their own complexity and interactive properties. Credit: Wyss Institute at Harvard University

(BOSTON) — DNA nanostructures assembled from shorter DNA sequences as building blocks have long sparked the imagination of bioengineers because their precisely programable size, shape, and functions could open up a plethora of non-biological and biological possibilities. If they could be built to be sufficiently large and complex, we could harness them to create highly programmable optical instruments, smart drug delivery tools, and objects that either perform functions of a living cell or instruct cell behavior in intricate ways, from the nanoscale up.

In the assembly of “DNA origami,” the most common nanofabrication strategy for such DNA nanostructures, a long DNA scaffold strand is folded is folded into a 3-dimensional object that is held in its desired shape by a multitude of complementary staple strands that “pull” the scaffold into the desired shape. However, the maximum size of a DNA origami object is limited by their scaffold strands, which become prone to shearing beyond a certain length. Researchers have tried to create larger structures by assembling single origamis together, but efficiently linking them to each other has been challenging. Thus far, these roadblocks have prevented the creation of DNA nanostructures above the sub-micron range, which would significantly expand their usability in real-world applications.

Now, a team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Dana-Farber Cancer Institute (DFCI) has found a solution to this problem. They extended their previously developed concept of “crisscross polymerization” to pre-fabricated highly diverse and stable DNA origami building blocks. This allowed them to assemble more than 1,000 DNA origami in a strictly seed-dependent fashion into the first multi-micron DNA megastructures with custom shapes and surfaces that can be fully addressed and patterned with functional molecules at the nanoscale. The “crisscross origami” method is published in Nature Nanotechnology.

We believe that crisscross origami is the biggest leap forward in the programmable self-assembly of complex shapes since the advent of DNA origami.

William Shih

“We believe that crisscross origami is the biggest leap forward in the programmable self-assembly of complex shapes since the advent of DNA origami. Efforts over the past fifteen years at the hierarchical assembly of non-identical DNA origami into larger structures have failed to proceed at anywhere near the efficiency of staple strands incorporating into a single DNA origami,” said Wyss Core Faculty member William Shih, Ph.D., who led the team. Shih co-leads the Wyss Institute’s Molecular Robotics Initiative and is also a Professor at Harvard Medical School (HMS) and DFCI.

Starting from a single DNA origami, which was developed in 2006, a doubling in size every two years would have predicted a thousand-origami megastructure such as the ones created in the new study to emerge by 2026. The invention of crisscross origami now has dramatically shortened this time-frame. “Our work sets the pace for advancing the complexity of DNA nanostructures ahead of a Moore’s-Law-style doubling every two years, and we envision that relatively simple extensions of our method should be able to fast-forward crisscross origami by several more doublings in the coming years,” Shih said.

From single DNA origami to multi-origami megastructures

Crisscross origami builds on the concept of “seed-dependent crisscross polymerization” that Shih’s team had developed in an earlier study. The researchers figured out a way to initiate the growth of DNA “ribbons” only in the presence of a pre-formed seed. Using this nanofabrication strategy, building blocks made from single strands of DNA called “slats” are programmed to bind first to the seed, and then to the steadily growing end of the ribbon by forming weak interactions with a large number of other slats. Each single interaction between an isolated pair of slats is too weak to sustain formation of a stable structure, such that ribbons will not form unless the seed is present.

By carefully tuning the strength of specific interactions between the slats, we achieved absolute seed-dependent growth of megastructures and leveraged that control to create the most complex multi-component origami structure ever demonstrated.

CHRIS WINTERSINGER, former graduate student mentored by Shih and now Program Manager at Speculative Technologies

In their new study, Shih and his team hypothesized that the principle of seed-dependent crisscross polymerization could be generalized to a significantly more complex building block: an already prefabricated DNA origami slat with a mass a couple of hundred times larger than that of a linear DNA slat sequence. “By carefully tuning the strength of specific interactions between the slats, we achieved absolute seed-dependent growth of megastructures and leveraged that control to create the most complex multi-component origami structure ever demonstrated,” said Chris Wintersinger, Ph.D., one of three co-first-authors, who performed his thesis work with Shih at the Wyss Institute and DFCI as a student of the John A. Paulson School of Engineering and Applied Sciences (SEAS). “With this method, we demonstrated complete growth of single micron-scale structures each composed from 1,022 different origami monomers. We see this as a path forward to self-assembling devices with levels of complexity that begins to rival that of systems we see in living cells.” Wintersinger now is Program Manager at Speculative Technologies.

DNA nanostructures grow up to become micron-scale megastructures
To demonstrate the functionalization capabilities of their crisscross origami approach, the team assembled megastructures in the shape of 2D sheets, attached a pattern of contrast agent molecules to select origami slats, which created the pattern of a happy face. Credit: Wyss Institute at Harvard University

As a resource for this study’s and future large-scale nanofabrication efforts, the researchers compiled a library of about 2,000 DNA strands that can be combined to assemble any of one quindecillion (a one followed by 48 zeros) distinct DNA origami slats, providing them with a vast design space. They fed their structural designs to a computational algorithm that selected binding sites for each slat from the strand library, and then used an acoustic liquid-handling robot to perform the fluid transfers necessary to make each origami slat building block. Besides the megastructures composed from many unique slats, Shih and his team also created periodic 1D ribbons and 2D sheets.

“With our micron-scale megastructures, we demonstrated that we can intricately pattern the megastructure surfaces with a diverse array of functional molecular ‘guests’ for future biological and non-biological applications,” said co-first author Dionis Minev, Ph.D. Minev, also a SEAS graduate and now a Staff Scientist at the Wyss Institute, teamed up with Wintersinger early in their graduate work to develop the crisscross-polymerization paradigm. They were joined by the third co-first-author Anastasia Ershova, a graduate student on Shih’s team. Separately, Minev and Ershova are pursuing seed-dependent crisscross polymerization of single-stranded DNA as a diagnostic approach at the Wyss Institute (Crisscross Nanoseed Detection).

Adding function to size

DNA nanostructures grow up to become micron-scale megastructures
Overview of crisscross assembly of DNA-origami slats (crisscross origami). The top row describes the composition of a DNA megastructure starting with a small “gridiron” seed structure to which a set of unique DNA origami slats is added. The middle row shows the breadth and relative scale of the megastructures created by the researchers using their crisscross origami approach. The bottom row shows DNA ribbons and sheets grown in one dimension (left) and two dimensions (middle), as well as an illustration of how origami slats can be assembled in an automated fashion by combining DNA strands from a strand library using a robotic liquid handler. Credit: Wyss Institute at Harvard University

To functionalize their megastructures, the researchers attached additional handle sequences to their origami slats that could accept guest molecules such as nano-sized contrast agents or fluorescent label strands. This enabled them to visualize their products not only using a common molecular-imaging technique called transmission electron microscopy (TEM), but also a different DNA nanotechnology-driven approach to high-resolution microscopy based on the concept of DNA-PAINT, which was developed by co-author and Wyss Core Faculty member Peng Yin, Ph.D. and his group. “To illustrate the functionalization capabilities of crisscross origami, we generated and visualized megastructures that displayed intricately patterned designs such as a jigsaw puzzle piece, a happy face, and the institutional logos for some of our affiliations,” added Minev.

Thinking ahead, the team envisions numerous applications for their novel nanofabrication approach. “Nanofabrication with crisscross origami unlocks a myriad of possibilities of how we can build better over larger distances with fewer error rates. The sheer size and functionalization capabilities of such megastructures could enable the development of new lens-like optical instruments with light-sensitive units arranged at the nanoscale and, excitingly, cell-sized architectures with the ability to control the behavior of cells,” said Ershova. Such large DNA megastructures, for example, could be used to instruct cells of the immune system in subtle, yet more effective ways to enable them to better fight tumors, or cells in injured tissues to go into repair mode.

“This fundamentally new approach of building functional objects using DNA as a programmable material with an unprecedented precision and into a size range that was not accessible before is a true milestone for DNA nanotechnology that opens up a plethora of new and exciting possibilities for us to interact with our physical and biological environments,” 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 Bioinspired Engineering at SEAS.

Other authors on the study are present and former Wyss Institute members Hiroshi Sasaki, Gokul Gowi, and Jonathan Berengut, and Eduardo Corea-Dilbert from DFCI. The study was funded by the Wyss Institute, National Science Foundation (DMREF award# 1435964, and award# CCF-1317291), Office of Naval Research (award# N00014-15-1-0073 and N00014-18-2566), and National Institutes of Health (NIGMS award# 5R01GM131401).

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