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.