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April 30, 2009

DNA Origami Self-Assembled Growth Details

This is a more detailed follow up to the original nextbigfuture article announcing the development of a method to grow DNA origami. This article discusses more details of the methods and error rates of what was done. This is a pathway to assembling larger complex molecularly precise structures.


Recently there was success with the algorithmic self-assembly of DNA tiles to grow DNA Origami.
Programmable DNA origami seeds were developed that can display up to 32 distinct binding sites and researchers demonstrated the use of seeds to nucleate three types of algorithmic crystals. In the simplest case, the starting materials are a set of tiles that can form crystalline ribbons of any width; the seed directs assembly of a chosen width with >90% yield. Increased structural diversity is obtained by using tiles that copy a binary string from layer to layer; the seed specifies the initial string and triggers growth under near-optimal conditions where the bit copying error rate is <0.2%. Increased structural complexity is achieved by using tiles that generate a binary counting pattern; the seed specifies the initial value for the counter. Self-assembly proceeds in a one-pot annealing reaction involving up to 300 DNA strands containing >17 kb of sequence information. In sum, this work demonstrates how DNA origami seeds enable the easy, high-yield, low-error-rate growth of algorithmic crystals as a route toward programmable bottom-up fabrication.


The 6 page paper is here



Programmingribbon width. (A–C)AFMimages of ligated ribbonsgrown
from seeds specifying 8-, 10-, and 12-tile-wide ribbons, respectively. (Scale bars:
1m.) (D, F, andH) Respective high-resolutionAFMimages of individual ribbons.
(Scale bars: 50 nm.) (E, G, and I) Distribution of ribbon widths in corresponding
samples of unligated ribbons (SI Text), given as the fraction of tiles found in
ribbons of a given width. Solid bars indicate samples annealed with seeds. n
41,718; 69,200; and 125,876 tiles, respectively. Dots indicate samples annealed
without seeds. n 23,524; 26,404; and 145,376 tiles, respectively. In each experiment, boundary and nucleation barrier tiles were at 100 nM each, and the
repeatable block tile concentrations were proportional to their use in ribbons of
the target width, i.e., 200, 300, and 400 nM, respectively. For samples with seeds,
each staple strand was at 50 nM, each adapter strand was at 100 nM, and the
origami scaffold strand was at 10 nM.








The effective nucleation of Variable-Width, Copy, and Binary Counter ribbons using information-bearing origami seeds points the way to reliable and programmable bottom-up fabrication of complex molecular structures. This success was based on several principles. (i) Each tile set was capable of generating an infinite variety of distinct structures, a precondition for programmability. (ii) A designed nucleation barrier prevented the spontaneous assembly of tiles in slightly supersaturated conditions, clearing the way for high-yield seeded growth with low error rates. (iii) Information contained in the seed was propagated, and sometimes processed, during crystal growth, enacting a simple developmental program. The system developed here has already been useful for growing DNA crystals with other algorithmic patterns. At the same time, this work uncovers several problems that must be solved to improve the technique. (i) The rate of copying errors that changed 1 to 0 was 5–10 times higher than errors changing 0 to 1, suggesting that modifying tiles by adding DNA hairpins as an AFM contrast agent significantly alters the crystal growth energetics. Alternative labeling methods could reduce the 1-to-0 copying error rate dramatically. (ii) Premature reversal errors and spurious nucleation errors could be reduced by adding an independent nucleation barrier on the other edge of the ribbon. (iii) Nucleation on the seed could occur more readily if the origami seed were redesigned to match the tile lattice spacing. (iv) Here, we used unequal tile concentrations to prevent excess tiles from forming undesirable side products. In contrast, theory predicts that error rates are lowest when the concentrations of all tiles are equal throughout the reaction. Low error rates and elimination of side products could be simultaneously achieved by purification of crystals before growth creates an imbalance in concentration, use of a chemostat, or design of a chemical buffer for tile concentrations (R. Hariadi, personal communication). (v) Implementing improved proofreading techniques should further reduce logical error rates and larger block sizes may reduce internal lattice defects. Finally (vi), aggregation of crystals must be reduced. Combined with the wealth of available chemistries for attaching biomolecules and nanoparticles to DNA, an improved system for seeded growth of algorithmic crystals could be a powerful platform for programming the growth of molecularly defined structures and information-based materials.

The artificial systems developed here fill the gap between the simple seeded growth of natural crystals and the sophisticated seeded growth of biological organisms. Some natural systems also occupy this gap: Similar phenomena—seeded growth of crystals with fixed thickness, variable thickness, combinatorial layering patterns, and even complex patterns derived from local interactions have all been observed or inferred in minerals such as rectorite, illite, kaolinite, barium ferrite, and mica.Within biology, centrioles that nucleate microtubles in the ‘‘9 / 2’’ arrangement to form cilia or flagella can be seen as information bearing seeds for molecular self-assembly. Thus, in addition to their technical relevance, the ability to study seeded growth processes using programmable DNA systems may open up new approaches for studying fundamental natural phenomena.


15 pages of supporting information describe how errors were reduced and various methods that were used.





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