The usefulness of DNA extends beyond carrying the hereditary material for living organisms. In the 1980s, researchers began to manipulate genetic strands—which are composed of four nucleobases (A, C, T, G) that form base pair bonds—to fold DNA (and, later, RNA) into predetermined structures. This includes scaffold-based DNA origami and DNA bricks. Such prototypes could eventually lead to nanoscale devices with real-world applications such as nanofabrication of molecular robots that deliver drugs upon contact with a target molecule; molecular instruments that precisely position molecules or trigger interactions; and molecular breadboards that organize hundreds of molecular components into specific arrangements with nanometer precision.

Until recently, DNA- and RNA-based structures have largely been limited to those built from multiple strands rather than single strands. To create such structures, researchers first synthesize a single-stranded DNA molecule called the scaffold and then add hundreds or thousands of individually synthesized shorter components. These bind to multiple locations on the scaffold, tying the molecule together and inducing folding. This complex assembly process makes the structures vulnerable to defects or missing strands, however, and necessitates time-consuming checks to ensure all pieces are correctly in place.

Now, as reported in a recent issue of Science, researchers have bypassed those limitations by creating stable, self-folding single-strand DNA and RNA origami that is both simpler and less prone to defects than earlier multi-strand products. “This is really a new way of making complex nanostructures with user prescribed geometries,” says Peng Yin, a professor of Systems Biology at Harvard Medical School and the Wyss Institute at Harvard University, and a co-author on the work. “We demonstrated that uni-molecular folding of a single strand without other components can be a general platform to produce complex structures.”

The concept of one long DNA or RNA strand whose sequence dictates its own folding first emerged in 2014, when researchers created A-form helices—one of the three biologically active double helical structures that DNA and RNA can adopt—from single-stranded RNA. The molecular design process was fairly complex, however, and sizes were limited to just 660 nucleotides.

Yin and his colleagues expanded on that earlier work by first identifying molecular structures that would allow their single-stranded structure to self-assemble in a precise order, preventing tangles and knots from forming. A web-based automated software they created based on those findings provides researchers with a general platform for producing complex structures from a single long strand.

“This is a very important paper, because it is one of the few that addresses the folding of nucleic acid strands while being aware not merely of energetics, but the topology itself, so as to avoid knotting,” says Ned Seeman, the Margaret and Herman Sokol Professor of Chemistry at New York University, a pioneer in the field of structural DNA nanotechnology, who was not involved in the research. “The algorithm is a major advance.”

Unlike multi-stranded DNA and RNA origami that require in vitro synthesis, the researchers verified in the laboratory that the single-strand folded into user-defined structures in vitro can be replicated in vivo by introducing the DNA sequences into bacteria, including E. coli. Producing molecules in this way would lower costs and chances of mistakes, the research team believes, as nanostructures could be cloned at high volume by replicating bacteria. Polymerase chain reaction amplification—a common technique used to amplify copies of a segment of DNA—was also demonstrated to reproduce the molecules.

In this first proof-of-concept study, the researchers were able to program protruding DNA loops that acted as handles for adding protein functional groups onto self-assembling large and single-strand DNA origami molecules of up to 10,000 nucleotides. They also created single-strand RNA nanostructures of up to 6,000 nucleotides—10 times the size and complexity of what was previously possible.

Erik Winfree, a professor of computer science, computation, and neural systems, and of bioengineering, at the California Institute of Technology, who was not involved in the work, describes it as “an incredibly elegant design approach for making single-stranded DNA and RNA origami, solving the problem of intrinsic ‘knotting’ of the strands that plagued previous thinking and coming up with a straightforward method for molecular design.” 

Yin and his colleagues imagine that future work will include creating single-stranded DNA and RNA nanostructures that could be filled with or attached to enzymes, fluorescent probes, metal particles, or pharmaceuticals, paving the way for a diversity of potential applications, from single-molecule tracking and drug delivery devices to components for nano-electronics.

As Yin says, “Essentially, once you have the ability to precisely control the structure and shape of a nanoscale particle and that particle can be added to other functional elements, there are endless possibilities.” 

Read the abstract in Science.