DNA Origami 68
FleaPlus writes "Caltech scientist Paul Rothemund has developed a new technique for designing and generating self-assembling 2D nanostructures out of DNA. To demonstrate the technique, which is reportedly simple enough that a high-schooler can design with it, Rothemund created patterns like smiley faces, text, and a map of the Americas. The technique might be useful for generating 'nanobreadboard' scaffolds for things like molecular-scale circuitry, protein-based factories, and quantum computers. Rothemund is currently working to extend the technique to 3D nanostructures."
Text from end of nature article (Score:5, Informative)
The scaffolded self-assembly of DNA strands has been used to create linear structures and proposed as a method for creating arbitrary patterns. But the widespread use of scaffolded self-assembly, and in particular the use of long DNA scaffolds in combination with hundreds of short strands, has been inhibited by several misconceptions: it was assumed that (1) sequences must be optimized20 to avoid secondary structure or undesired binding interactions, (2) strands must be highly purified, and (3) strand concentrations must be precisely equimolar. These three criteria are important for the formation of many DNA nanostructures and yet all three are ignored in the present method. For example, M13mp18 is essentially a natural sequence that has a predicted secondary structure which is more stable (lower in energy) than similar random sequences (Supplementary Note S8). Further, stocks of staples each contained a few per cent truncation products, stock concentrations were measured with at least 10% error, and staples were used successfully at stoichiometries that varied over an order of magnitude.
I suggest that several factors contribute to the success of scaffolded DNA origami (even though the method ignores the normal, careful practices of DNA nanotechnology). These are (1) strand invasion, (2) an excess of staples, (3) cooperative effects and (4) design that intentionally does not rely on binding between staples. Briefly (details are given in Supplementary Note S9), strand invasion may allow correct binding of excess full-length staples to displace unwanted secondary structure, incorrect staples, or grossly truncated staples. Further, each correct addition of a staple organizes the scaffold for subsequent binding of adjacent staples and precludes a large set of undesired secondary structures. Last, because staples are not designed to bind one another, their relative concentrations do not matter.
The method presented here is easy to implement, high yield and relatively inexpensive. Three months of effort went into the design program. In addition, each structure required about one week to design and one week to synthesize (commercially); the mixing and annealing of strands required a few hours. The greatest experimental difficulty was acquiring high-resolution AFM images, typically taking two days per structure. For rigid designs using circular scaffolds (rectangles with patterns, three-hole disks, and sharp triangles), yields of qualitatively well-formed structures were at least 70%. A better understanding of folding will depend on less-destructive imaging and quantification of small ( 15 nm) defects. A possible objection to the routine use of the method is the potential cost of staples; unlike the scaffold, staples cannot be cloned. However, unpurified strands are inexpensive so that the scaffold constitutes 80% of the cost, even when using a 100-fold excess of staples (Supplementary Note S10).
I believe that scaffolded DNA origami can be adapted to create more complex or larger structures. For example, the design of three-dimensional structures should be accessible using a straightforward adaptation of the raster fill method given here. If non-repetitive scaffolds of megabase length can be prepared, micrometre-size origami with 20,000 features may be possible. However, the requirement for unique sequence information means that the method cannot be scaled up arbitrarily; whenever structures above a critical size or level of complexity are desired, it will therefore be necessary to combine scaffolded DNA origami with hierarchical self-assembly, algorithmic self-assembly, or top-down fabrication techniques.
An obvious application of patterned DNA origami would be the creation of a 'nanobreadboard', to which diverse components could be added. The attachment of proteins, for example, might allow novel biological
Two other links. (Score:4, Informative)
His personal page [caltech.edu] is promising more details by last thursday... (oops). He's out to lunch right now (OK: Supper), so It'll be at least a couple of hours before he gets the update installed (he has been given the heads up).
Re:Stuff I didn't get from TA (Score:4, Informative)
1. How does the template interact with the DNA to cause self-assembly in the desired pattern?
I'm not sure exactly what you mean by template, but essentially what is mixed in solution is one very long strand of DNA and many very small fragments (staples). DNA has 4 bases (A, G, T, C) that bind specifically in AT and GC pairs. When bases are combined into strands they have complementary sequences that will preferentially bind to them, such as AGTT binding to TCAA. In this technique the staples bind to the long strand in such a way as to make it fold a specific way, causing it to automatically assume the desired shape. I didn't read the actual Nature article, but it seems likely that the staples serve to stabilize long portions and force 180 degree turns at the necessary locations on the long DNA strand.
2. If I throw RNA in with the object, can the structure reproduce?
No. Look at DNA replication on wikipedia; RNA doesn't really have much to do with replication.
3. Since these are all based on a single gene, they all code for the same protein, right?
No. These are not based on genes. These are arbitrary sequences of DNA designed and produced synthetically. They do not code for proteins.
4. How could these structures be used for molecular computing? (the article hints at it; I want details).
If we can create any arbitrary 2D structure with 6 nm resolution, this is a major leap over conventional lithography. This means that a 2D design could be made using DNA and then used as a guide for carbon nanotubes or some other technology, vastly improving resolution.
Other DNA nanotech labs (Score:2, Informative)
Ned Seeman [nyu.edu]
William Shih [harvard.edu]
Eric Winfree [caltech.edu]
John Reif [duke.edu]
Re:Stuff I didn't get from TA (Score:4, Informative)
1. How does the template interact with the DNA to cause self-assembly in the desired pattern?
The thing to understand is that the template is the DNA. DNA binds to other DNA rather specifically, with the A's binding to the T's and the C's binding to the G's. Normally, there's two strands, with one strand containing the binding partners of the other strand. However, in this case, there'll be only one long strand and a bunch of other "staples". The long strand will bind to itself and also the "staples" to form this structure. The self-assembly is induced because it's energetically favorable - the A's "want" to pair with T's, etc. When DNA is heated to around the boiling point of water, however, all of these hydrogen bonds between strands are broken (but, importantly, the strands themselves remain intact). So now a bunch of single-stranded DNA is floating around, but when it's cooled, it assembles into structures. Normally, two DNA strands that are complemenatry would anneal, but in this case, the scientist designs the strand to bind to itself instead. Because it binds to itself in specific places, it forms a predictable structure. In this case, the scientist also used little bits of additional DNA to hold the structure together.
RNA often forms itself into this sort of secondary structure in nature, but that's typically boring stem-loop structures. In this case, the scientist takes our existing knowledge of nucleotide secondary structure and tried to make his own more interesting secondary structure, to great success.
2. If I throw RNA in with the object, can the structure reproduce?
Sadly, it's not as simple as "throwing RNA in there", there needs to be certain enzymes and also the complementary strands of all the DNA in order to work properly. However, it is likely that with the correct mix of enzymes, DNA primers, etc that these things could reproduce themselves, although they'd have to be reassmbled afterwards. I don't think they can replicate themselves in their assembled form.
3. Since these are all based on a single gene, they all code for the same protein, right?
I must've missed the part about these coding for genes at all. DNA doesn't have to code for any genes, it can be just DNA that sits around and assembles itself into structures, which I think is what this is.
4. How could these structures be used for molecular computing?
Unfortunately, I don't know much about molecular computing, but DNA is a small, predictable, availible substance that we are rapdily getting better at manipulating. Because of this, it's probably the best bet for near-future nanotech, possibly including molecular computing.
If you are on a university or library connection, you can probably check out the main scientific article here [nature.com], which has lots of cool figures and is probably a lot more informative than what I've said. If you're not at a place that can access it, you can find the publication itself anywhere that gets Nature (probably your public library and almost certainly your nearby university). It'd be in the 16 March 2006 issue.
How the DNA origami works (Score:5, Informative)
The Virus strand:
The virus strand serves as the basic starting material for the origami. It's a single stranded, 7000 base long piece of DNA from a virus that attacks bacteria. There are only two reasons the virus strand is used:
1. It is nonrepeating. This is important because every group of 8 bases have a pretty much random sequence of DNA, and can therefore serve as a unique address for a particular position along the length of the virus strand (you get 4^8 = 65536 possible addresses). Thus, in this way, you can address ~1000 distinct points along the length of this viral DNA.
2. It's readily available. Since you can harvest the DNA from the virus, it's cheap to produce. In fact, this strand is commercially available.
DNA staples:
To actually make the virus fold into position, you need several hundred pieces of DNA to serve as staples that stitch together specified positions along the virus strand. Each staple is 32 bases long. Say that you want to stitch together positions A, BC, and D on the virus strand. You then make a staple whose first 8 bases are complementary to those at position A on the virus, whose next 16 bases are complementary to position BC, and whose final 8 bases are complementary to D. The DNA staple will then bind to those positions in solution and staple positions A, BC, and D together into a rigid, tightly packed structure.
You can buy any 32 base long sequences of DNA that you specify from the internet, so getting several hundred distinct strands is no big deal.
Okay, now how do I make a shape?
Think of how you would draw a smiley face with a CRT screen. Your computer has the outlines of the smiley face in memory, and raster-fills the shape. In the case of a virus origami, you first specify the outlines of the shape, then you raster-fill it with the virus strand by running the virus strand side to side from top to bottom. You then figure out all the staples you need to hold your raster-filled shaped together. Finally, you get the sequences, buy them over the internet, throw them together with the virus strand in a solution, and wolah, you get the world's smallest smiley face.
Is this important?
Paul Rothemund may get a trip to Stockholm some day.