Developmental biology - DNA|
How To Tie A Knot In DNA
A method for coaxing single-stranded DNA into complex 2 and 3D knotted structures...
Knots are indispensable tools for sailing, fishing and rock climbing. But tying a knot in a strand of DNA - measuring billionths of a meter in length - requires enormous patience and highly specialized tools. Hao Yan PhD, a researcher at Arizona State University (ASU) is a practiced hand in this delicate and exotic field. He operates at the crossroads of nanotechnology and fine art. In his new research appearing in the journal Nature Communications, Yan and his colleagues describe how they coax segments of single-stranded DNA into complex 2 and 3D knotted structures.
Their research represents an important advance in the fast-paced field of DNA nanotechnology, where molecules are configured to make a vast array of tiny devices. Among these are miniscule robots, photonic (light) applications, drug delivery systems, computer logic gates, as well as in diagnostic and therapeutic applications.
"Knotted DNA structures demonstrate unprecedented topological [the mathematics of stretching, twisting, crumpling and bending] complexity. It is not only amazing - but surprising, that single-stranded DNA and RNA can thread through its own chains and form highly knotted structures." Hao Yan PhD, Director, Biodesign Center for Molecular Design and Biomimetics, ASU, Tempe, Arizona, USA.
Bringing DNA into the fold
In nature, strings of nucleic acids provide the code needed to make complex proteins. This basic pattern is the underpinning of all life on earth. Taking advantage of DNA's simple base-pairing properties, it is possible to design structures that self-assemble in the lab. This method applied to both single-stranded and double-stranded DNA has resulted in complex nanostructures with increasing sophistication.
While DNA origami has made startling advances, one technical aspect has been difficult. Creating complex knotted DNA structures in a predictable and programmable way. However, new work establishing precise design rules is beginning to see single-stranded segments of DNA (or RNA) form with 1800-7500 nucleotides, into knot-like nanostructures with 9 to 57 crossings - where the DNA strands weave in and out of its own length.
The group went on to demonstrate that nucleic acid nanostructures can replicate and amplify under laboratory conditions and in living systems. These knotted structures, like those that Yan fabricated, have correlates in the natural world. They are seen when a DNA sequence is copied into messenger RNA. They can also occur in the genomes of phages - viruses that infect bacterial cells. Nevertheless, the construction of molecular knots at the nanometer scale, displaying well-defined and consistent geometry, requires enormous control and precision.
As it happens, nucleic acids like DNA are ideal for the design and synthesis of such molecular knots.
The study is innovative in the field of DNA origami as it uses nucleic acids like DNA and RNA to fold and self-assemble into complex structures using the strict regimen of DNA's 4-letter alphabet:
• Cytosine (C) always pairs with Guanine (G)
• Adenine (A) always pairs with Thymine (T)
Previously, lengths of double-stranded DNA have been used for nanoscale constructions, with the addition of short pieces or "staple strands" to fasten structures together. The new study instead uses a single length of DNA designed to wrap around itself in a precise, pre-programmed sequence of steps. Once the knotted DNA nanostructures successfully assemble themselves, they are imaged using atomic force microscopy. Careful calculation allowed researchers to optimize the folding pathways and produce the highest yield for each synthetic structure. The use of single- rather than double-stranded DNA allowed the structures to be produced in abundance and at much lower cost.
A single-stranded approach opens the door for the design of nanoarchitectures with specific functions, where desired attributes are selected through repetitive processes of refinement. The approach outlined in the new study provides for the design of molecular structures of increased size and unprecedented complexity, paving the way for advances in nanophotonics, drug delivery, cryo-EM analysis and DNA-based memory storage.
Designer DNA and RNA
The initial knot designs Yan and his colleagues developed threaded a single strand of DNA or RNA through itself 9 times, demonstrating their method is capable of producing intricate geometric shapes. The design was expanded with a technique known as cryogenic transmission electron microscopy. The rules of folding are based on the number of (1) crossing points, (2) the length of DNA and (3) the number of base pairs in a desired structure. Following this strategy, the team created more complex DNA knots with increased crossings. A DNA knot structure with 57 crossings was successfully assembled, with lower yield and less precision. When the crossing number was increased to 67, the yield significantly dropped and the resulting structures had more errors in assembly.
The study reports the largest DNA knots assembled, were formed with up to 7.5k bases, and featuring the most complicated twists and turns with up to 57 crossings. These single stranded DNA sequences can be mass produced in living cells for greater efficiency and at lower cost. Ultimately, DNA nanostructures with diverse functions may be able to be formed within cells and will be pursued in future work by the group.
Molecular knots represent one of the most extraordinary topological structures in biological polymers. Creating highly knotted nanostructures with well-defined and sophisticated geometries and topologies remains challenging. Here, we demonstrate a general strategy to design and construct highly knotted nucleic acid nanostructures, each weaved from a single-stranded DNA or RNA chain by hierarchical folding in a prescribed order. Sets of DNA and RNA knots of two- or three-dimensional shapes have been designed and constructed (ranging from 1700 to 7500 nucleotides), and they exhibit complex topological features, with high crossing numbers (from 9 up to 57). These single-stranded DNA/RNA knots can be replicated and amplified enzymatically in vitro and in vivo. This work establishes a general platform for constructing nucleic acid nanostructures with complex molecular topologies.
Xiaodong Qi, Fei Zhang, Zhaoming Su, Shuoxing Jiang, Dongran Han, Baoquan Ding, Yan Liu, Wah Chiu, Peng Yin and Hao Yan.
This research was supported by Office of Naval Research grant N000141512689 to H.Y. and National Science Foundation grants 1360635, 1563799, and 1334109 to H.Y. W.C. and Z.S. gratefully acknowledges funding support from National Institutes of Health (NIH P41GM103832 and P50GM103297). P. Y. acknowledges funding support from Office of Naval Research (N000141612410)..
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Design of single-stranded DNA (ssDNA) or RNA knots. The formation of a knot involves the threading of two ends of a loop-stem structure following a pathway similar to that shown in b. Credit: Nature Communications.