Session 5: Nanofabrication via Structural DNA
Transcript of Part 1: Nanofabrication via DNA Origami
00:00:07.15 My name is William Shih. 00:00:08.23 I'm an associate professor of Biological Chemistry 00:00:11.15 and Molecular Pharmacology 00:00:12.23 at Harvard Medical School, 00:00:14.11 Dana-Farber Cancer Institutes, 00:00:16.06 and the Wyss Institute for Biologically Inspired Engineering. 00:00:19.22 It's my pleasure to share with you today 00:00:21.25 some recent technical advances 00:00:23.13 in the field of structural DNA nanotechnology, 00:00:26.29 from my laboratory and those of my colleagues. 00:00:30.04 We're all familiar with the biological role of DNA 00:00:33.02 as an information repository, 00:00:35.22 principally for coding for protein sequence 00:00:38.06 and for regulation of protein expression. 00:00:41.08 And I'm not going to speak at all about that today. 00:00:44.08 Instead, I'll be talking about using DNA itself 00:00:47.12 as a building material 00:00:49.02 and harnessing that in order to us 00:00:51.04 to construct nanoscale objects. 00:00:53.17 For example, shown here is an electron micrograph 00:00:55.22 of actin filaments 00:00:57.19 that are about 7 nanometers (nm) in diameter. 00:01:01.04 And below, we can see this peculiar 00:01:03.10 Pac-Man-shaped object. 00:01:05.06 This is a structure that was built entirely from DNA 00:01:08.24 and we designed it for the purpose 00:01:10.27 of taking bite-size chunks 00:01:12.15 out of the actin filament. 00:01:14.10 Well, this project hasn't yet been successfully, 00:01:17.06 however hopefully this image drives home 00:01:19.05 the complementarity between the dimensions 00:01:21.16 of our designed DNA nanostructures 00:01:24.08 and biological macromolecular complexes. 00:01:28.01 So I've been working in this field 00:01:29.11 for over a decade now, 00:01:31.10 and I'm continually surprised 00:01:33.05 by advances in the field. 00:01:34.25 I have these preconceived notions of the limitations of DNA, 00:01:37.21 and they're always shattered 00:01:38.25 by the latest new discovery. 00:01:40.19 And today I'm going to be sharing with you, 00:01:42.10 in the first two sections, respectively, 00:01:44.25 two recent developments 00:01:46.16 that enable us to design and assemble 00:01:48.20 DNA nanostructures of the size and complexity 00:01:51.09 of this object shown here, 00:01:52.18 about 30 nm in diameter. 00:01:55.14 One of the most important goals for DNA nanotechnology 00:01:58.18 is to self-assemble objects 00:02:02.06 of ever-increasing complexity over time. 00:02:05.06 For example, is it possible that, 00:02:07.29 within the next decade or two, 00:02:09.14 that we can self-assemble objects 00:02:11.12 that are let's say a thousand times as complicated, 00:02:14.12 that have a thousand times as many unique components, 00:02:17.05 as the object shown here? 00:02:20.02 A second question is: 00:02:21.12 what are these things good for? 00:02:23.02 And in the third part of this lecture, 00:02:24.09 I'll be discussing some applications in my laboratory 00:02:27.11 as tools for molecular biophysics 00:02:29.22 and tools for future therapeutics 00:02:32.02 where we think these objects might prove useful. 00:02:35.00 We're inspired from natural systems, 00:02:37.02 we know that they can carry out many amazing behaviors: 00:02:40.18 they can build, they can adapt, 00:02:42.15 they can heal, they can reproduce. 00:02:45.05 And these are capabilities that human technology 00:02:47.04 struggles to reproduce on any kind of length scale, 00:02:50.00 but what's especially remarkable 00:02:52.00 is the ability of life to carry these out 00:02:54.08 on the molecular scale. 00:02:55.23 So, for example, here on the left 00:02:57.16 is a picture of a ribosome, 00:03:00.03 and this is of course a machine that's about 25 nm in diameter 00:03:04.05 that takes information encoded in messenger RNAs 00:03:07.10 and then translates that into 00:03:08.21 a specific sequence of amino acids, 00:03:10.26 to produce a polypeptide. 00:03:13.18 Quite amazing machine. 00:03:16.06 On the right, we have the T4 bacteriophage, 00:03:19.06 that's a bit larger in length scale. 00:03:21.02 We can see that the viral capsid itself -- head capsid -- 00:03:23.26 is about 100 nm in diameter, 00:03:25.17 so it looks like a little nanoscale hypodermic syringe 00:03:27.19 that docks onto the surface of its bacterial hosts 00:03:30.26 and then injects its DNA cargo into the cell 00:03:33.25 against an osmotic pressure gradient. 00:03:37.05 Of course what makes this all possible 00:03:39.00 is that living systems have invented molecular manufacturing 00:03:43.08 and they've come up with a very robust and clever way of doing this. 00:03:46.08 They, of course, synthesize these biopolymers 00:03:49.07 -- they can be polypeptide chains, polynucleotide chains -- 00:03:52.11 that then self-assemble into the desired structure. 00:03:56.25 Now, if somebody asked you about ten years ago, 00:04:00.08 "Would it be possible to generate an object 00:04:03.02 of this complexity using some kind of human-based technology?" 00:04:06.00 most people would have been skeptical. 00:04:08.17 And yet I'm going to show you a new technology, 00:04:10.27 DNA nanotechnology, developed in the last... 00:04:14.04 especially with advances from the last ten years, 00:04:16.24 that now make it possible for us to self-assemble, 00:04:19.12 in a programmable way, 00:04:21.12 structures of the same kind of complexity 00:04:23.12 as you see here. 00:04:24.26 Not yet of the functional complexity, 00:04:27.01 but nevertheless we think this is an encouraging first step 00:04:30.02 because along the pathway to this kind of functionality, 00:04:33.16 we first need to master structural complexity. 00:04:39.21 We're going to be using DNA as our building material, 00:04:43.04 and we know that DNA, 00:04:45.22 very similar to proteins and other macromolecules from life, 00:04:48.26 are very complicated molecules. 00:04:51.07 There's many different atoms, 00:04:52.24 but it turns out the key point for DNA nanotechnology 00:04:55.26 is that the robust base-pairing properties of DNA 00:04:58.20 allow us to abstract away those chemical details, 00:05:01.26 which is going to make the act of 00:05:04.00 designing the nanostructures much simpler. 00:05:06.23 And in fact there's only three characteristics of the DNA 00:05:09.19 that we need to remember 00:05:10.28 for the purpose of DNA nanoconstruction. 00:05:13.13 One, is that it's a ladder with antiparallel strands. 00:05:17.24 Secondly, there's a right-handed twist for B-DNA, 00:05:21.04 and we need to know that the twist 00:05:22.23 is around 10.5 basepairs per turn. 00:05:25.15 It turns out we can switch that around a little bit. 00:05:28.29 And finally we need to know that 00:05:31.11 A pairs with T, 00:05:32.16 C pairs with G, 00:05:34.08 and anytime you deviate from that pairing, 00:05:36.20 you're going to destabilize the structure. 00:05:39.25 And it's because the propensity of DNA 00:05:42.17 to form this very regular structure, 00:05:44.21 enforced very strictly according 00:05:46.08 to this Watson-Crick base pairing, 00:05:48.03 that gives us its power in being able to 00:05:50.19 generate these large structures with very little design work. 00:05:54.10 The father of the field of DNA nanotechnology 00:05:56.23 is Ned Seeman at NYU. 00:05:58.28 He invented this field about thirty years ago. 00:06:02.10 His training is as a crystallographer, 00:06:04.07 and the way he came up with the idea is as follows: 00:06:06.16 he was sitting in the campus pub one day, 00:06:08.20 just drinking his beer, and suddenly 00:06:10.24 what popped into his head was this woodcut 00:06:12.29 from M.C. Escher. 00:06:15.00 He had been collaborating with some of his friends 00:06:17.14 on DNA Holliday junctions, 00:06:19.28 and he had this eureka moment: 00:06:21.11 why not replace the flying fish 00:06:23.23 with DNA Holliday junctions? 00:06:25.24 The notion was that if you could rationally design 00:06:28.18 a porous crystal out of DNA, 00:06:30.24 and then he could take the target protein he was interested in, 00:06:33.02 and then dock that into each unit cell 00:06:35.02 into a stereotyped orientation, 00:06:37.13 then he would be able to impose that crystalline order 00:06:40.01 on the target protein, 00:06:41.19 and therefore make the X-ray crystallography problem easier 00:06:45.02 for these large macromolecules 00:06:46.20 that are otherwise difficult to crystallize. 00:06:50.20 He's been working on this problem for over thirty years, 00:06:53.00 it's an important goal, 00:06:54.01 and he's made some interesting progress, 00:06:55.25 I'll have more to say about that in the third segment. 00:06:59.26 But in the meantime, he's had some interesting 00:07:01.25 landmark successes. 00:07:03.25 So the first really noteworthy advance that he reported 00:07:07.13 came out in 1992 or so in Nature, 00:07:10.14 so this is a DNA cube 00:07:12.20 where each edge of the wireframe cube 00:07:14.20 is two turns of a double helix. 00:07:16.27 Each face is a circular strand of DNA 00:07:20.12 and the entire object has dimensions 00:07:22.09 of about 10 nm. 00:07:25.02 So the first time I think people saw this they thought, 00:07:27.15 "Wow, this is really cool, 00:07:29.21 but that doesn't look like biology to me." 00:07:32.22 And what I hope convince you today 00:07:34.17 is that this is in fact an extremely powerful technology. 00:07:38.12 Yes, it's fun, 00:07:39.18 but it's actually potentially very useful as well, 00:07:42.01 for many different applications. 00:07:48.06 Of course if we're building from DNA strands 00:07:50.19 and we're just making double helices 00:07:52.03 then that's boring. 00:07:53.15 The power of DNA nanotechnology 00:07:55.08 is that we can build with branched junctions. 00:07:57.27 With the previous example, the cube, 00:07:59.13 each one of those vertices 00:08:00.25 is a three-branch junction. 00:08:02.23 But it turns out the most powerful motif 00:08:04.21 so far in structural DNA nanotechnology 00:08:07.13 has been a four-branch junction; 00:08:09.09 a Holliday junction. 00:08:11.03 So on the upper left here we have a schematic 00:08:14.07 using simple letter notation of the strands, 00:08:17.22 so you can see the cyan strand starts 00:08:19.22 5'-CCGG, goes to it's 3' and, 00:08:23.05 and if you look closely at this you can see that 00:08:24.23 there's four different sequences 00:08:27.06 and they have the proper sequence complementarity 00:08:29.17 in order to generate a Holliday junction 00:08:31.20 that actually is immobile. 00:08:33.14 It can't branch migrate due to its sequence. 00:08:36.23 And we know from structural studies 00:08:38.15 that this object likes to stack into two double helices 00:08:42.12 that are connected at a joint, 00:08:44.18 and it turns out this is really the building block 00:08:46.10 that's been the most fruitful for DNA nanotechnology. 00:08:49.29 So the idea is as follows: 00:08:51.23 if you only have one Holliday junction, 00:08:53.28 now you have two helices that can wobble around. 00:08:56.13 In order to fix those two helices 00:08:58.01 to make a rigid building block, 00:08:59.22 what we do is we simply introduce 00:09:01.15 a second Holliday junction downstream, 00:09:04.00 and now when we fix those two double helices 00:09:06.04 with two Holliday junctions 00:09:07.17 we have that rigid building block that we want, 00:09:09.23 now with four sticky ends. 00:09:12.10 The next step is to build two versions of this building block. 00:09:15.24 In this example, we have a red one 00:09:17.24 and we have a blue one. 00:09:21.02 And we designed the sticky ends with the following 00:09:22.06 complementarity in this example. 00:09:24.17 So let's say we make the sticky ends 00:09:26.05 on the upper right-hand side of the red block, 00:09:29.02 and we make that compatible with 00:09:31.19 the lower left-hand side of the blue block. 00:09:35.00 And so on and so forth, 00:09:36.10 in order to create this kind of checkerboard fashion, 00:09:38.24 hopefully you can see that we would be able to self-assemble 00:09:41.15 these two bricks into an infinite two-dimensional lattice, 00:09:44.12 as shown below. 00:09:46.25 I don't have the experimental images for this, 00:09:48.29 but suffice it to say that this method actually worked. 00:09:52.24 Quite amazing - you can design a two-dimensional crystal. 00:09:56.29 The step after this would be to say, 00:09:58.21 "Well, instead of two bricks, two tiles, 00:10:02.02 what if I had ten tiles, 00:10:03.23 or what if I had 100 tiles? 00:10:05.05 Can I now make non-periodic structures 00:10:07.11 that are highly complex, 00:10:09.07 just with self-assembling tiles?" 00:10:10.26 And unfortunately, nobody has really 00:10:11.25 demonstrated this method, 00:10:14.12 extending this particular method to 100's of tiles, 00:10:17.09 although what I'll show you shortly is one method, 00:10:20.14 DNA Origami, that can achieve this kind of complexity, 00:10:23.03 and in the second segment, 00:10:25.06 something called single-stranded bricks, 00:10:26.17 that can do something very similar 00:10:27.29 to what I just described. 00:10:30.23 The method of DNA Origami 00:10:33.03 is a particular flavor of structural DNA nanotechnology. 00:10:36.05 It was developed by Paul Rothemund at Caltech, 00:10:38.18 he published this in 2006, 00:10:40.28 and the basic idea is as follows: 00:10:43.22 so imagine you have a long single strand of DNA, 00:10:46.16 the 7000 base genome of the M13 bacteriophage, 00:10:50.05 that's the gray strand in this animation. 00:10:52.21 We know what the sequence is, 00:10:54.01 and based on that known sequence, 00:10:56.01 we chemically synthesize 100's of short oligonucleotides 00:10:59.11 that are 20-60 bases long 00:11:01.17 that are programmed by Watson-Crick complementarity 00:11:04.16 to pinch that long strand 00:11:06.06 into a parallel array of helices, 00:11:08.17 after heating everything up to about 65°C 00:11:11.24 and then cooling down to room temperature 00:11:13.28 over the course of an hour. 00:11:16.11 At the end of the assembly, 00:11:18.05 you end up with this parallel array 00:11:20.09 of double helices 00:11:22.06 where adjacent double helices are held together 00:11:24.01 by these Holliday junction crossovers 00:11:25.28 that I described to you a couple slides ago. 00:11:27.27 So this is a half-crossover 00:11:29.11 and then here we have a full DNA crossover. 00:11:34.10 Importantly, what you just saw was an animation, 00:11:36.18 not a simulation. 00:11:37.24 In fact, we have a very poor understand 00:11:39.15 of the order of events of folding these objects, 00:11:42.19 we just know that if we program them 00:11:44.18 in a way where all of the scaffold ends up basepaired 00:11:47.24 to staple strands, 00:11:49.12 then we have an extremely high probability 00:11:50.27 of forming the desired structure. 00:11:53.04 So it's a very active area of research for us to 00:11:55.01 try to understand better the mechanism of folding, 00:11:58.03 and we hope that will actually help us to design more 00:12:01.01 complex structures in the future. 00:12:03.16 So Paul Rothemund used this method in 2006 00:12:05.29 to make structures such as this 00:12:07.24 'disc with three holes' 00:12:09.16 that has dimensions of about 100 nm x 100 nm x 2 nm, 00:12:13.17 this is an atomic force micrograph. 00:12:15.17 The example in the upper left-hand corner represents, in size, 00:12:18.14 just part of the upper lip of the object. 00:12:21.04 So this is quite large by macromolecular standards. 00:12:23.29 It's like we have two ribosomes worth of molecular Silly Putty 00:12:27.07 that we can mash into any desired 00:12:29.16 two-dimensional cookie cutter shape. 00:12:32.02 One of the very interesting things that he pioneered 00:12:34.12 was that he developed a way to 00:12:36.22 make this DNA origami 00:12:38.11 where he made each one of the staple strands 00:12:40.17 in two different flavors. 00:12:42.11 So one flavor 00:12:44.01 just made the structure as you saw. 00:12:46.17 The second flavor had the identical sequence, 00:12:49.01 but had a surface feature, 00:12:50.19 a dumbbell that's sticking out of one of its ends. 00:12:53.24 And so what that means is any time he used the original flavor 00:12:57.02 and he added it to the folding mix, 00:12:58.27 then you'd get a plain vanilla DNA Origami surface 00:13:02.05 at that location. 00:13:04.13 But then if he replaced that sequence 00:13:05.27 with the longer sequence, the one with the feature, 00:13:08.07 now you get that same shape, 00:13:09.25 but a bump over that feature. 00:13:12.22 And in that way, 00:13:13.27 he conceived that this rectangular DNA Origami 00:13:16.21 could be treated as a molecular breadboard, 00:13:18.28 where let's say it has 200 different positions, 00:13:21.17 we can decide at each position 00:13:23.06 whether we want to create a bump or have no bump. 00:13:26.18 In effect we have something that's like a bitmap, 00:13:28.28 and we can create new patterns 00:13:30.13 simply by repipetting different patterns 00:13:32.18 of the no-bump and plus-bump strands 00:13:35.01 for each one of the locations. 00:13:36.16 So for example, here we can see that he's designing 00:13:39.03 something that will say 'DNA' 00:13:41.01 and have a little picture of DNA. 00:13:42.20 These structures actually become very sticky at the ends 00:13:44.09 because they have lots of blunt ends, 00:13:46.08 and then they'll make a continuous ribbon 00:13:48.14 that says 'DNA'. 00:13:50.19 You can see that he made a map of the Americas. 00:13:53.14 He's a very humble guy, 00:13:55.01 so he apologized to the rest of the world 00:13:56.13 for stopping at the Americas, 00:13:57.25 but DNA is a little bit expensive, 00:14:00.14 so he stopped at the moment. 00:14:02.26 Maybe by now he's made the rest of the world. 00:14:06.20 And you can program them to link up in specific ways, 00:14:10.29 and in that way you can self-assemble 00:14:12.25 two-dimensional crystalline objects. 00:14:16.02 So what about getting to three dimensions, as I alluded? 00:14:18.28 Well, we can get our initial inspiration 00:14:21.07 from macroscale paper Origami, 00:14:23.14 where we're quite familiar 00:14:25.17 that if we fold flat paper in many ways 00:14:28.11 we can get quite intricate three-dimensional shapes. 00:14:30.29 So this is the famous crane. 00:14:34.00 And if you're really diabolical, like Robert Lang, 00:14:38.11 you might note that if you can fold these papers 00:14:42.02 in especially intricate ways, 00:14:44.13 then you can make incredibly complicated objects, 00:14:48.18 that we can see some examples of here. 00:14:51.18 Now, nothing I'm going to show you with DNA 00:14:53.15 is as complicated as this, 00:14:55.05 but again, as I mentioned, 00:14:56.13 one of our goals is to scale to ever-increasing complexity, 00:14:59.08 so we hope that someday we actually can 00:15:01.20 self-assemble DNA into objects of this kind of complexity. 00:15:07.00 So that group in Denmark that I just mentioned, 00:15:09.10 of Andersen, Jørgen Kjems, Kurt Gothelf, 00:15:12.23 they were able to design that M13 00:15:15.08 to fold into six different sheets, 00:15:18.13 and then they programmed those six sheets to fold up 00:15:20.18 into a three-dimensional box 00:15:22.00 with a hollow inside. 00:15:23.24 They designed a lid that can open in response 00:15:25.20 to some kind of molecular key. 00:15:27.26 So this was the first example of 00:15:30.02 a three-dimensional hollow DNA Origami. 00:15:35.18 So where my group wanted to contribute 00:15:37.14 was to make solid three-dimensional Origami structures, 00:15:41.02 and the idea is as follows: 00:15:44.00 so first of all, we know that we can 00:15:46.10 curl up DNA due to the helicity of the DNA helices, 00:15:49.16 and I'm going to go through a little thought experiment 00:15:51.17 just to give you a flavor of what this is about. 00:15:53.25 So here we have, on the far left, 00:15:55.25 three double helices that are arranged 00:15:57.13 into a little DNA Origami. 00:15:59.06 You can see, if you look closely, 00:16:00.25 they're connected by those Holliday junction crossovers 00:16:03.00 to keep the helices parallel. 00:16:06.14 And in this arrangement it's making a flat sheet of three helices. 00:16:10.12 So now imagine what would happen 00:16:12.11 if we moved these crossovers on the top 00:16:15.21 two base pairs to the left. 00:16:18.16 Then that's going to move that double helix 00:16:20.12 behind the plane of the page. 00:16:22.15 And likewise, if we move those two crossovers 00:16:24.14 two base pairs to the right, 00:16:26.09 that's going to move that double helix 00:16:27.29 in front of the plane of the page. 00:16:30.00 And the take-home message here is that 00:16:31.12 simply by shifting around the position of those crossovers 00:16:34.23 with respect to each other, 00:16:36.18 we can achieve curvature of these DNA Origami sheets 00:16:40.05 along the axis of the double helices. 00:16:42.21 So that's the first key. 00:16:45.00 So now let's extend that and build an actual 00:16:46.24 solid 3-dimensional structure. 00:16:49.00 So here we have another representation of a DNA Origami 00:16:51.03 where each one of these cylinders 00:16:52.19 represents one of those double helices, 00:16:54.15 so it's similar to the example in the upper left, 00:16:56.16 but now just rotated into this orientation. 00:17:00.00 So this would represent the pattern of the scaffold 00:17:02.12 running through those helices, 00:17:04.05 but for the purpose of this explanation, 00:17:06.09 I'm going to leave that invisible. 00:17:08.15 It's there, but I'm just not going to talk about it, 00:17:10.18 that or the staple strands. 00:17:12.15 And so what we're going to do is we're going to 00:17:14.08 shift around the position of those crossovers 00:17:16.24 so now these helices no longer prefer to be planar, 00:17:19.25 but instead prefer to curl up 00:17:22.25 into some kind of specific geometry. 00:17:25.14 And in this example what we're doing is 00:17:27.08 we're trying to curl up the structure into a corrugated S shape. 00:17:32.04 Furthermore, anywhere where we have the orange 00:17:34.29 that touches the white sheet that touches the blue sheet, 00:17:37.13 we're routing those staple strands through those interfaces. 00:17:40.15 So for example, we might have a staple strand 00:17:42.09 that starts 7 base pairs on this helix, 00:17:44.22 and then goes 7 base pairs here, 00:17:46.12 7 base pairs, 7 base pairs, 7, 7 base pairs. 00:17:50.04 And in that way, if the structure forms the way we intend it to, 00:17:53.03 it should be highly crosslinked 00:17:55.17 by these staple strands that are traversing the different helices. 00:17:59.26 So it looks good on paper, okay, 00:18:03.03 what happens in the test tube when we tried it? 00:18:05.19 And perhaps we can say, 00:18:07.18 "Of course, when we threw all the strands together 00:18:09.16 and tried to fold the object, then it didn't work." 00:18:12.24 We got a pile of molecular spaghetti 00:18:14.24 that we could see under the electron micrograph. 00:18:18.12 But we didn't want to give up, 00:18:20.04 and eventually Hendrik Dietz in the group 00:18:22.08 came up with a key insight, 00:18:24.01 which is it's not that these 3-dimesional objects 00:18:26.13 now are unstable thermodynamically, 00:18:28.28 simply they're more difficult to achieve kinetically. 00:18:32.23 And so what we found is that we could only get appreciable yields 00:18:34.28 of these objects 00:18:36.27 if we folded them instead of for an hour, 00:18:39.04 from 65°C to room temperature, 00:18:40.27 if we folded them for more like a week, 00:18:43.15 then we could start to get appreciable yields 00:18:45.16 of the objects ranging from 10-50% yield. 00:18:50.13 So we can see here one of the objects 00:18:52.16 that was built by Shawn Douglas. 00:18:54.10 Instead of 3 layers, it was with 10 layers. 00:18:57.26 And then we have the electron micrographs below. 00:19:00.29 We can see that we get a close resemblance 00:19:03.02 between what we observe in the electron micrograph 00:19:05.07 and the projection orientations 00:19:07.28 of our designed structure. 00:19:12.05 This is work that we published back in 2009, 00:19:14.22 in the meantime, our group and others have been hard at work 00:19:17.11 trying to improve the method. 00:19:18.26 So the important thing here was that 00:19:20.06 we could get something to fold at all, 00:19:22.08 and now we're trying to get better yields, 00:19:24.11 improve the folding times. 00:19:26.01 So there have been a couple of important discoveries since then. 00:19:28.21 One has come from Hendrik Dietz's lab in Munich, 00:19:31.20 where they've discovered that these structures 00:19:33.16 tend to have a favored temperature 00:19:35.20 at which they fold faster than the other temperatures. 00:19:37.27 So instead of spending the same amount of time 00:19:39.28 at 65°C down to room temperature, 00:19:43.10 for example this structure maybe folds faster at 50°C. 00:19:48.11 So what they found is if they do most of their folding at 50°C 00:19:51.13 then they can get it fold 00:19:53.01 maybe an order of magnitude faster, 00:19:55.25 which makes a lot of improvements 00:19:58.28 for our lives as scientists designing them, 00:20:00.19 they also suffer less thermal damage with a slower folding ramp. 00:20:04.28 We've also learned some details about 00:20:06.26 how to design the strands, the crossovers, 00:20:09.12 the breakpoints, 00:20:11.01 that I don't have time to go into in this presentation, 00:20:13.24 but I encourage you to look at some of our publications 00:20:16.00 if you want to see the latest discoveries 00:20:18.01 in how to make this process work better. 00:20:22.23 So now I'm going to go through a panel, 00:20:24.21 a gallery of different objects built using this method 00:20:27.17 by our laboratory to give you a flavor of the generality of the method. S 00:20:31.20 o the example on the top is what I just showed you, 00:20:33.28 we call it the "Monolith", it was built by Shawn Douglas. 00:20:37.08 You might say that it looks a little bit like a nanoscale crystal, 00:20:40.23 honeycomb array crystal, 00:20:43.06 but it's important to keep in mind that every element 00:20:45.23 of the object is associated with a unique sequence 00:20:48.25 and therefore is independently addressable. 00:20:51.24 This is quite different from most nanoparticles 00:20:53.22 that we see in synthetic nanotechnology today. 00:20:58.27 The example on the bottom was built by Franziska Graf, 00:21:01.15 we call it the "Genie Bottle". 00:21:02.29 We called it that because one version, 00:21:04.29 not shown here, 00:21:06.15 we only folded part of the M13 scaffold 00:21:08.15 and the rest of it was coming out of the lip 00:21:10.00 of the object kind of like wisps of smoke. 00:21:13.13 These are all 20 nm scale bars. 00:21:18.10 So here again, 20 nm scale bars, 00:21:20.20 on the left is an object built by Shawn Douglas, 00:21:22.26 we call it the "Square Nut". 00:21:24.28 It has a 7 nm hole in the middle, 00:21:27.23 it has a front end and a back end, 00:21:30.16 and if we make the sticky ends on the front end compatible 00:21:32.14 with the sticky ends on the back end, 00:21:34.07 then we can self-assemble filaments 00:21:36.24 that are somewhat reminiscent of 00:21:39.05 actin filaments and microtubules, 00:21:40.29 although in this case they don't yet demonstrate any dynamics. 00:21:43.23 They're just equilibrium formation of these long polymers. 00:21:48.07 On the right is an object built by Tim Liedl, 00:21:50.12 we call it the "Railed Bridge". 00:21:52.22 Again, every cylinder is one double helix, 00:21:54.22 and we can see as we go through cross-sections of the object 00:21:57.08 we have a different arrangement of double helices, 00:21:59.22 and we can understand from this example 00:22:02.20 that it is kind of analogous to sculpture. 00:22:06.02 That you could imagine the sculptor begins 00:22:08.01 with a solid block of marble, 00:22:10.02 in our case these parallel arrays of double helices, 00:22:12.28 and in design space we're chipping away 00:22:14.20 at that solid block to achieve whatever 3-dimensional structure 00:22:17.25 we actually want in relief. 00:22:20.15 Once we have our final design, 00:22:22.01 then what we're doing is we're compiling that 3-dimensional structure 00:22:26.07 into a series of DNA strands 00:22:28.12 that are going to self-assemble with the M13 scaffold 00:22:30.27 into that object. 00:22:36.00 Here's an object built by Björn Högberg 00:22:38.05 when he was in the group, 00:22:39.11 we called it the "Slotted Cross", 00:22:41.03 I'll have more to say about this object in the next slide. 00:22:43.20 This is another crossed object we called 00:22:45.24 the "Stacked Cross", built by Hendrik Dietz. 00:22:48.24 Again, these are all 20 nm scale bars. 00:22:50.19 This one looks a little bit like stacked molecular celery. 00:22:54.20 We even designed a little molecular cavity on the top 00:22:57.17 where we initially imagined we could host protein guests 00:23:00.00 on the inside of that cavity. 00:23:02.21 So let's take a closer look at that "Slotted Cross" 00:23:05.08 from Björn Högberg. 00:23:07.05 So here what he's done is he's generated an animation 00:23:10.25 where he's stylized the routing of the scaffold strand 00:23:13.25 through the structure. 00:23:15.18 It's designed as an "H-domain" and an "O-domain", 00:23:20.14 and the middle of the H-domain is designed 00:23:22.13 to pass through the middle of the O-domain 00:23:24.14 and it's all folding from just one long M13 scaffold. 00:23:28.24 I was quite amazed that this folded at all, 00:23:32.01 but the yields are not so great at the moment, 00:23:34.09 just a few percent. 00:23:38.25 So now what I'm doing is I'm zooming in 00:23:40.28 on what we call the strand diagram 00:23:42.22 that describes the blueprint of the object. 00:23:45.04 It's like we take all the helices 00:23:46.22 and then we splay them out 00:23:48.05 onto a 2-dimensional surface. 00:23:50.13 And in this case the blue represents that M13 scaffold strand 00:23:54.19 and those colored strands represent the staple strands. 00:23:57.17 And this part of the object is the upper left-hand corner 00:24:01.04 of the H-domain. 00:24:03.03 And so if you look closely you can see that the staple strands, 00:24:06.00 what they're doing is they're binding to part of the scaffold strand 00:24:08.12 and then they're crossing over to a different part of the scaffold strand 00:24:11.15 to pull those components together 00:24:13.23 to make the 3-dimesional shape. 00:24:16.19 We can zoom out, 00:24:18.07 and then here you can get an appreciation 00:24:20.26 that it is kind of like a blueprint. 00:24:22.19 You can make out which part is the H-domain 00:24:25.03 , which part is the O-domain, 00:24:27.02 and if you look closely you can actually see 00:24:29.15 where the H-domain and O-domain are being connected 00:24:31.16 by that long scaffold strand. 00:24:38.08 All of the examples that I've shown you so far 00:24:40.07 have been built using this honeycomb lattice paradigm 00:24:45.10 where we're using these corrugated sheets. 00:24:47.29 It turns out that it more naturally fits 00:24:50.07 the preferred twist of DNA 00:24:53.01 at 10.5 basepairs per turn, 00:24:55.13 but it turns out we can also self-assemble these 00:24:57.26 DNA sheets in a square lattice format. 00:25:03.10 The only proviso is that now we have to 00:25:05.29 underwind the DNA to 10.67 basepairs per turn, 00:25:10.05 which is slightly disfavored. 00:25:12.06 And, quite interesting, 00:25:13.22 what happens is the structure will still form, 00:25:15.27 but it then compensates 00:25:17.22 by having a global supertwist 00:25:20.11 in the right-handed direction. 00:25:21.26 So it's quite analogous to how plasmid DNA, for example, 00:25:25.15 will have a right-handed supertwist 00:25:27.11 when it's underwound, as we find in most cells. 00:25:34.13 One very important development in the field 00:25:37.24 is software with a graphical user interface 00:25:39.26 to make it accessible to people who are outside the field, 00:25:42.13 but also just to make the process faster, 00:25:45.09 more robust and convenient, 00:25:47.01 for experienced practitioners. 00:25:49.12 So for this really powerful software suite 00:25:52.06 called "cadnano", 00:25:54.05 we owe our thanks to Shawn Douglas, 00:25:55.22 he developed this software when he was a graduate student in my group, 00:25:58.18 now he's an assistant professor at UCSF, 00:26:01.24 at the time of this filming. 00:26:03.14 So I encourage you to check out the software, 00:26:06.01 he's continually improving it, at cadnano.org. 00:26:09.10 And what we can do, now again with the graphical user interface, 00:26:12.21 within an hour or so, 00:26:14.03 we can design different shapes 00:26:15.21 and then compile that into the sequence of DNA strands 00:26:18.20 that can self-assemble into that object. 00:26:25.07 What if you wanted to build larger structures? 00:26:28.08 Well, the most obvious idea is to 00:26:30.13 just get more parts. 00:26:32.00 So you can remember as a kid, 00:26:34.15 the first time you got a Lego set it was enthralling, 00:26:37.19 but then about two hours later, 00:26:39.07 you now were hungry for additional Lego pieces. 00:26:42.10 So that's the big drive for our field: 00:26:44.07 can we get more Lego pieces into the structure? 00:26:46.21 But in the meantime we can do other things 00:26:48.19 that will allow us to get a little bit bigger. 00:26:50.16 So one example is just to build with wireframes, 00:26:52.21 that have high strength-to-weight. 00:26:54.24 So in this example what we've done 00:26:56.10 is we've added staple strands that fold that M13 scaffold 00:26:59.18 into this wireframe structure. 00:27:03.09 Each one of these struts in this example 00:27:04.28 is a 6-helix DNA nanotube, 00:27:07.12 and then we designed sticky ends such that they're compatible, 00:27:10.21 and we can get this structure, 00:27:12.27 this Z-shaped structure, 00:27:14.17 to fold into a double triangle, 00:27:17.09 with now 10 of these 6-helix bundle termini, 00:27:20.17 each with a unique set of sticky ends. 00:27:23.15 In this example, what we did is we programmed 00:27:25.13 3 separate double triangles to form 00:27:27.11 in 3 separate test tubes, 00:27:29.15 and we programmed it to form this network on the bottom. 00:27:32.22 This is a Schlegel diagram, 00:27:34.08 and for those of you who might recognize this, 00:27:36.18 you might see that this is actually a Schlegel diagram 00:27:38.23 for a wireframe icosahedron. 00:27:41.25 This object has an overall diameter of about 100 nm, 00:27:45.18 each one of the struts has a length of about 45 nm. 00:27:49.25 And here on the lower left-hand corner 00:27:51.19 we can see an animation, macroscale animation, reenactment, 00:27:55.05 of the self-assembly of these double triangles 00:27:57.13 into a wireframe icosahedron. 00:28:07.24 What we find is that this process works in the test tube as well - 00:28:09.27 no hands required. 00:28:12.01 So again, what we do is we fold each of the double triangles 00:28:14.12 in three separate test tubes, 00:28:16.02 we then mix them together to form the desired wireframe object. 00:28:21.01 So let's take a look using electron microscopy. 00:28:23.18 So here we see with a 1 micron (um) scale bar, 00:28:26.01 we see a bunch of objects 00:28:28.06 that seem to have about the right size, about 100 nm in diameter. 00:28:31.07 There's aggregates as well, 00:28:32.19 so the self-assembly's not perfect, 00:28:35.14 but we're glass-half-full kind of folk, 00:28:38.03 we're encouraged by something that works 00:28:40.05 even partially. 00:28:42.04 So now we've zoomed in, you have a 500 nm scale bar, 00:28:45.00 and we can tell that there's some kind of wireframe action going on. 00:28:48.11 Zoom in some more, 00:28:49.23 now we have a 200 nm scale bar, 00:28:51.28 and it's starting to look like the wireframe structure 00:28:55.00 that we imagined. 00:28:56.27 Of course you have some mis-assemblies as well. 00:28:59.26 And then now if we go to the highest effective magnification 00:29:01.23 for this negative stain method, 00:29:03.17 100 nm scale bar, 00:29:05.11 we can see the objects, in fact, 00:29:06.29 they look like they have lots of these triangular faces, 00:29:09.18 they look like they have 5-fold vertices. 00:29:14.14 And we're able to make an object that now is 00:29:17.09 something like five times the mass of a ribosome, 00:29:20.17 it has overall dimensions the size of a medium size virus. 00:29:24.16 And this is all just powered 00:29:25.18 by Watson-Crick base pairing: 00:29:27.05 A pairs with T, 00:29:28.12 C pairs with G. 00:29:30.12 It's remarkable that we can push it this far, 00:29:32.25 ~but we're greedy and we dream about 00:29:34.13 being able to extend this to objects 00:29:36.17 that are a thousand times more complex 00:29:39.09 or even more than that some day. 00:29:43.26 Another kind of wireframe structure 00:29:45.25 from macroscale engineering 00:29:47.15 that inspired us are these floating compression sculptures 00:29:50.06 from the artist Ken Snelson. 00:29:53.10 And the idea here for these sculptures 00:29:55.11 is that you have these beams, 00:29:57.09 that are bearing compression, 00:29:58.14 that aren't touching each other directly, 00:30:00.09 but instead they're connected by cables 00:30:02.08 that are bearing tension. 00:30:03.20 And if you wire this up in the correct way, 00:30:06.15 then it's a balance between the 00:30:08.00 tension of the cables 00:30:09.20 and the compression of the beams, 00:30:11.00 and you end up with an object that has 00:30:12.03 high strength-to-weight 00:30:13.17 and has other interesting features. 00:30:15.09 For example, if those cables have some elasticity, 00:30:17.21 then if you put a global force on the object, 00:30:19.20 then it will deform, 00:30:21.17 and every individual strut will rearrange in 3-dimensional space. 00:30:26.05 When you now relieve that stress, 00:30:27.26 then it'll bounce back to the original shape. 00:30:30.05 So we wanted to see if we could implement this 00:30:32.07 using DNA Origami. 00:30:34.24 This is work that was led by Tim Liedl and Björn Högberg 00:30:37.22 when they were in the group, 00:30:38.16 in collaboration with Don Ingber. 00:30:40.26 So what they did was 00:30:42.09 to design the staple strands to fold this M13 scaffold 00:30:46.17 into 3 different struts, 00:30:49.11 each of the struts in this case is 13 helices. 00:30:52.05 It's actually grabbing 3 separate segments 00:30:54.11 of that scaffold in order to make each one 00:30:56.21 of those 13-helix struts. 00:30:59.28 So we again add everything together, 00:31:01.06 heat it up, cool it down, 00:31:03.00 and remarkably enough you can form structures 00:31:05.16 like this in the test tube. 00:31:07.27 In fact, we started to play games about 00:31:09.21 looking at how much stress we could put the objects under 00:31:13.05 and have them still fold. 00:31:14.19 So what happens is that you have these single-stranded 00:31:16.16 DNA elements that are acting like entropic coils 00:31:19.08 - they're exerting tension. 00:31:21.06 And if we simply design those cables to be shorter, 00:31:25.02 have fewer number of bases, 00:31:26.28 then it's going to exert a larger force 00:31:30.01 over the same design distance between the two compressed elements. 00:31:34.18 And what we found by continually shortening these cables 00:31:38.15 is that we could self-assemble the structures 00:31:40.18 up to about 14 piconewtons of force, 00:31:43.09 that was the calculated force for the shortest cable 00:31:45.20 that were able to self-assemble the objects. 00:31:48.25 In other words, we're able to self-assemble these DNA objects 00:31:52.02 against twice the force that can be generated by 00:31:55.14 powerful cytoskeletal motors such as kinesin or myosin. 00:31:59.09 This is all powered by just DNA base pairing. 00:32:03.17 We believe that these kind of structures may prove 00:32:07.26 useful for applications in tissue engineering 00:32:10.01 and regenerative medicine. 00:32:11.23 So of course cell biologists have noted for a while now 00:32:15.11 that cells, especially going through development, 00:32:17.29 can communicate with their outside environment, 00:32:20.28 with each other, using mechanics. 00:32:22.26 So they might pull on the extracellular matrix 00:32:25.00 and that extracellular matrix may pull back, 00:32:28.10 and you might have, 00:32:29.29 by introducing deformations into the extracellular matrix 00:32:33.03 or within the cytoskeleton of the cell, 00:32:35.25 you can trigger biochemical events. 00:32:37.29 So we envision a day where we can 00:32:39.11 use these kind of DNA nanostructures 00:32:41.15 that can deform in response to some kind of mechanical stress 00:32:45.10 and then translate into a biochemical event, 00:32:47.05 it could be release of a growth factor, 00:32:49.27 or maybe it could involve catalysis of some kind of chemical reaction. 00:32:54.14 So we believe that this could be useful for regenerative medicine. 00:33:00.02 So the last thing that I'd like to show you for this section 00:33:03.04 is work from Hendrik Dietz, 00:33:05.13 Shawn Douglas assisted on this work. 00:33:07.18 Everything that I've shown you thus far has involved double helices 00:33:10.18 that are straight. 00:33:12.14 And Hendrik wanted to ask the question, 00:33:14.07 "Could you build structures, curved structures, 00:33:16.24 where the helices now are following an arc, 00:33:18.28 instead of going straight?" 00:33:21.02 And the basic strategy for implementing this is as follows: 00:33:24.04 so here we have, again every cylinder represents a double helix, 00:33:27.21 these planes that are slicing through the double helices 00:33:31.06 represent the positions at which those crossovers are occurring. 00:33:34.14 So it turns out in this example 00:33:36.01 they're only occurring every 7 basepairs. 00:33:40.21 And he asked the question, well, 00:33:42.09 what would happen if he replaced 00:33:44.09 the double helical segments on the top, so the orange segments, 00:33:48.21 with shorter double helices 00:33:50.14 that only have 6 base pairs between planes. 00:33:53.23 And what if he replaced the helices on the bottom, 00:33:56.08 the blue ones, 00:33:57.25 instead of 7 basepair segments, 00:33:59.21 he had 8 basepair segments. 00:34:01.25 So mechanically, now on the top, 00:34:04.06 those elements are going to be under tension 00:34:07.09 because you have less material in the same amount of space, 00:34:09.15 they're going to be stretched out. 00:34:12.08 The helices are the bottom are going to be under compression, 00:34:14.29 because we now just stuffed more material 00:34:17.09 into the same amount of space. 00:34:19.24 And the system is under stress 00:34:21.07 and so it's going to relax, of course, by bending. 00:34:24.26 So this is the way to relieve that tension on the top 00:34:27.10 and compression on the bottom. 00:34:29.08 Does this actually work 00:34:30.06 when we attempt this in the test tube? 00:34:32.08 And the answer is yes. 00:34:34.08 So Hendrik implemented this, 00:34:36.02 using an 18 helix DNA bundle 00:34:39.13 that's illustrated on the upper left-hand side. 00:34:43.20 And so what he did was he had a stereotyped straight region, 00:34:46.18 these white regions, 00:34:48.09 and then he had an experimental region 00:34:50.19 that's indicated here in red. 00:34:52.24 So that's where he's going to be introducing 00:34:54.13 those longer and shorter elements 00:34:56.10 to induce the bending of the structure. 00:34:59.12 You can see for the control you get this nice rigid straight object. 00:35:04.18 So what happens when he introduces 00:35:06.17 some small number of shorter strands 00:35:08.21 in the double helices on top, 00:35:10.12 and then longer ones on the bottom. 00:35:11.27 He could get a reliably predicted 00:35:13.21 30 degree arc at that position. 00:35:16.27 If he has roughly twice the number of perturbations, 00:35:19.20 then you can get to a 60 degree angle. 00:35:24.14 Kept on going, you get 90 degree, 00:35:26.00 you can get a 120 degree angle, 00:35:27.19 that's quite remarkable. 00:35:30.03 This is now getting down to a 10 nm radius of curvature. 00:35:32.29 But then he kept on going, 00:35:35.07 and he found he could go all the way 00:35:36.21 to 180 degrees in this example. 00:35:39.29 So this is something that has a 6 nm radius of curvature 00:35:42.20 , it's comparable to the tightness of wrapping of 00:35:45.15 DNA double helices around histones in a nucleosome. 00:35:49.12 So in that case that's powered by protein-DNA interactions, 00:35:51.27 in our case this is powered by 00:35:54.04 DNA base pairing interactions. 00:35:56.22 So here what we have is an animation prepared by Shawn 00:35:59.28 that explains the bending principle. 00:36:02.20 So again, what we're doing is 00:36:03.28 we're introducing more basepairs, 00:36:08.00 or long double helices on the left, 00:36:10.05 and then shorter ones on the right. 00:36:14.05 And you can see a little graph on the lower left-hand side 00:36:16.15 that tells us how many basepairs per turn 00:36:18.13 that we have 00:36:20.05 for each of these different elements. 00:36:21.24 And at the most extreme example we're actually 00:36:23.17 getting 15 basepairs per turn on the left, 00:36:26.21 which is severely underwound, 00:36:29.02 and only six basepairs per turn on the right, 00:36:31.11 which is severely overwound. 00:36:33.19 And I was quite flabbergasted 00:36:35.27 that it should be possible for us to 00:36:37.26 torture DNA to this extent. 00:36:40.10 Now in fact once you get to those extremes, 00:36:41.27 our folding yields do start to go down, 00:36:44.05 so we can see that we're at the edge 00:36:46.02 of what we can do to DNA, 00:36:47.29 but still it's quite remarkable that DNA is so robust, 00:36:52.14 that the 10.5 basepairs per turn 00:36:54.26 is simply what it prefers to do, 00:36:56.11 but if you put enough stress on it, 00:36:58.01 you can make it do things that deviate 00:36:59.29 from that ideal by quite a bit. 00:37:06.20 So Hendrik and Shawn 00:37:08.14 now used the method to make a variety of different structures. 00:37:11.09 So on the upper left-hand corner 00:37:13.10 we have a 6-helix DNA bundle 00:37:15.03 that's folded into a series of 00:37:18.18 180 degree arcs of increasing radii of curvature, 00:37:21.27 so you make a spiral. 00:37:24.22 On the lower left-hand corner we have an object 00:37:27.02 that's programmed to self-assemble 00:37:29.07 into a beach ball, out of 6-helix bundles. 00:37:34.01 You can see objects that are making concave triangles, 00:37:37.19 this is designed by Shawn Douglas. 00:37:39.22 And then here we have those 00:37:41.19 120 degree arcs that are repurposed, 00:37:44.13 so we made sticky ends on the two ends 00:37:46.15 of this little boomerang to be complementary, 00:37:49.11 so that you can have three identical versions of them 00:37:51.27 will come together to make a larger triangle. 00:37:56.07 So in conclusion, 00:37:57.10 hopefully I've persuaded you that DNA Origami 00:38:00.09 is a highly versatile method 00:38:01.28 for building both 2-dimensional 00:38:03.19 and 3-dimensional structures of 00:38:06.06 quite remarkable complexity, 00:38:07.25 about twice the mass of a ribosome. 00:38:10.22 Where we're moving to next is to try to build structures 00:38:13.13 that are more complicated. 00:38:14.26 You might wonder what's preventing us from building 00:38:16.20 something 1000 times larger already, today. 00:38:19.12 And the main problem is that 00:38:20.21 we have errors in the self-assembly. 00:38:24.00 For example, for one of these objects, 00:38:26.09 we might have a yield, in the best cases, 00:38:29.06 75% of so, 00:38:30.22 which might sound pretty good. 00:38:32.11 But now if you wanted to build an object that's 1000 times bigger, 00:38:36.00 then you might argue that the probability, 00:38:38.02 if you just mixed these things together, 00:38:39.21 1000 of them together, 00:38:40.27 the probability that none of the 1000 00:38:43.13 would have any defect, 00:38:44.28 would be 0.75^1000, 00:38:47.08 which if you do the math, that's basically zero. 00:38:50.00 So there's a lot of activity in the field 00:38:52.12 trying to improve the fidelity of this self-assembly, 00:38:55.26 other methods like hierarchical self-assembly, 00:38:57.28 error correction, 00:38:59.08 that'll allow us to scale amount complexity 00:39:01.16 and build really very complicated objects of the future.