[00:00] Thank you so much again for joining Will. [00:02] And it's always a pleasure to have you at these workshops. [00:05] And we're very excited for your talk. [00:06] Thanks. [00:07] I'm going to be telling you about our work [00:09] in crisscross polymerization, led by talented junior [00:13] investigators. [00:14] Two of them are here, Chris Wintersinger and Anastasia [00:17] Irsheva. [00:19] Third key contributor, Dio Mena, was a former foresight fellow. [00:22] And then we also have Jie Dong, who just took a faculty [00:25] position in Wuhan. [00:26] And I'll be telling you about a reminder of what [00:28] I talked about last year of building [00:31] what we call megastructures from building blocks that [00:34] are DNA origami. [00:36] And then I'll be sharing very briefly some exciting advances [00:39] that Anastasia led in trying to get exponential growth [00:43] from single-stranded slots. [00:46] And the starting point is thinking about DNA origami. [00:49] And what I love the most about DNA origami [00:51] is that you have absolute control [00:53] over the number of structures that form [00:56] based on the number of scaffold strands that you add. [01:00] But what if you wanted to build structures that [01:02] are far bigger than a scaffold? [01:06] Because the problem is that with DNA origami, half the mass [01:09] has to be the scaffold. [01:10] And so that's the problem we set out to solve. [01:12] Why would you want to do that? [01:13] Well, we can envision a future where [01:15] we can program the self-assembly of squishy robots [01:18] the size of a cell that might be a million times bigger [01:22] than an individual DNA origami. [01:23] We don't have a scaffold half the size of a cell. [01:27] Contrarily, another point is we would [01:29] like to be able to build ultra-sensitive enzyme-free [01:32] diagnostics. [01:33] We're trying to convert a single analyte [01:36] into vast polymerization of single-stranded building blocks [01:39] into double-stranded products. [01:40] So build a lot of material for amplification. [01:43] And again, this is something beyond the capability [01:45] of conventional DNA origami. [01:48] So I'm going to introduce this concept of crisscross [01:51] polymerization, where the goal is [01:52] to program some kind of building block that's [01:56] thermodynamically specified to assemble into desired shape, [01:59] but is completely blocked kinetically from ever doing so, [02:03] except when you add a small seed that is much tinier [02:06] than the final assembly. [02:08] And so the basic ingredient is specifications [02:10] you have a building block through these elongated slats [02:13] with many specific glues on them. [02:15] In this case, these are DNA origami slats. [02:17] Each one of these long things is one DNA origami. [02:19] It has 32 sequence-specific glues on it. [02:23] We program them so that they can make interactions [02:25] with each other at roughly 90 degrees. [02:28] And it's programmed in the final assembly to have 32 neighbors. [02:32] But it can only make one contact with any one neighbor. [02:37] Importantly, we operate this at a temperature [02:39] where you need close to half of the bonds, or 16 in this case, [02:43] for stable assembly. [02:44] So if you only made eight bonds or nine bonds, [02:47] it'll transiently come together. [02:49] And then it'll fall apart. [02:51] And that's really the key of designing a process that [02:54] will never ever start kinetically, [02:56] even though it's favored thermodynamically. [02:58] So we can imagine a growth trajectory where we have two [03:00] slats come together. [03:01] The energy deficit is represented. [03:03] One represents the cost. [03:04] One unit is the cost of fixing one of those building blocks [03:08] into exactly the right position entropically. [03:11] And so just bringing two things together, [03:12] it's making only one bond. [03:14] Well, that's not favored. [03:15] So that's very unstable. [03:18] But what's even more unstable is trying to add two more slats. [03:21] Because every time you add another slat, [03:22] you're making fewer than those 16 bonds. [03:24] So you're just writing up the energy landscape, [03:28] climbing Mount Improbable, you might call it, [03:31] to make successively more preposterously unlikely [03:34] structures. [03:35] Until you finally get to this structure [03:37] we call the critical nucleus, where finally once you achieve [03:39] this mountain peak, you can start adding building blocks [03:42] where each added building block is making 16 bonds [03:45] and therefore is stable. [03:47] But the situation is to get to this peak, [03:49] we need to climb an energy barrier of 16 units. [03:53] In other words, the entropic price of fixing 16 building [03:56] blocks in exactly the right orientation [03:58] without anything to hold them together. [04:00] Which will basically, you could fill the ocean with these things [04:02] and then wait the age of the universe. [04:04] It's just not going to happen as long as you're [04:05] close to that reversible temperature. [04:07] And then that will buy us the opportunity [04:09] to control this process by putting in a little deus [04:13] ecmakina, a single denier origami seed that's [04:15] using very strong bonds to position those first 16 [04:19] horizontal structures. [04:21] It pays that entropic price and now the process [04:23] can go off and running, where we can imagine starting [04:26] with this bidirectional growth front, [04:28] we're adding 16 slats at a time, and then we [04:31] can towel around into some desired structure. [04:34] And so we can see a transmission electron micrograph here [04:36] of an object approaching a micrometer in scale [04:39] with 192 origami. [04:41] We published this work in Nature and Nanotechnology [04:43] and the station designed this beautiful cover. [04:46] So these structures are made from over 1,000 denier origami. [04:50] Each are different. [04:51] Overall dimensions of two microns by two microns. [04:55] And right now we're working on trying [04:57] to extend this to make it in more order magnitude bigger, [05:00] or maybe two orders magnitude bigger, try to make 3D. [05:02] I don't have time to go into those details right now. [05:04] Happy to talk about it later. [05:06] I just want to briefly point out I stuck this [05:08] into one of the working documents, [05:09] this idea of how to use crisscross for implosion. [05:12] So I'm not really going to talk about it here. [05:14] I'm just going to briefly mention that the point of this [05:17] is that it's how do you build pre-assembled three [05:20] to five nanometer building blocks into much bigger [05:22] structures if that's what you want. [05:24] This is agnostic about how you make those smaller building [05:26] blocks. [05:27] It's really about that next step. [05:29] And so now I'd like to briefly talk about crisscross [05:32] with single-stranded building blocks [05:33] for the purpose of signal amplification for diagnostics. [05:38] And you can repeat this crisscross process [05:41] with the same advantages of kinetic blockade, [05:43] but with now tiny, tiny building blocks that [05:45] are just oligonucleotides. [05:47] And the oligonucleotides are just binding to each other [05:49] with half a turn. [05:51] That's one binding site. [05:52] And the same process where you specify that at equilibrium, [05:56] all the binding sites are occupied [05:57] by a bunch of different binding partners, [06:01] but that kinetically it never gets off the starting point [06:04] because of this entropic barrier. [06:06] So you're writing up this climbing non-improbable, [06:09] and it's so rare to get to the peak [06:10] that this process basically never happens. [06:13] But now we have single-stranded building blocks, [06:14] so the assembly can happen much more quickly. [06:16] We have these building blocks at very high concentration. [06:19] And again, if we introduce a seed that somehow [06:21] is linked to the analyte that we're trying to detect, [06:23] in this case, Sedinia ergami, then we [06:25] can trigger the process in a controlled fashion. [06:27] So it nucleates the assembly, and then it grows. [06:30] We have ideas of how to convert any analyte into such a seed. [06:34] Happy to talk about it later. [06:36] And really, the brand new idea that Anastasia [06:39] has been developing that we're very excited about [06:41] is how do we actually get this to grow not just linearly, [06:45] but exponentially? [06:46] It turns out linear growth is too slow in order [06:48] to get truly in-fold amplification. [06:50] And so the scheme, we came up with a strand displacement [06:53] based scheme in order to cut these thick filaments. [06:55] And that might sound very difficult, [06:57] but we figured out a scheme that works. [07:00] And it's based on this notion of toe-hole mediated strand [07:02] displacement that many of you might be familiar with, [07:04] that you can think of this as how do we [07:06] break the blue and the black strands apart? [07:09] That's kind of cutting this into two pieces. [07:11] And the way that we do this is by introducing [07:14] a single-stranded toe-hold that the displacing strand can [07:16] grab onto to nucleate, undergo branch migration, [07:19] and kick the black off. [07:20] This is how we split the blue and black into two strands. [07:24] And then in this case, in the middle, [07:26] is taking this analogy with one of our crisscross structures. [07:29] So here we're asking the green strand in this middle thing [07:33] to come in and displace the top slot away. [07:36] And so it kind of comes in. [07:37] And I'm not going to describe this in great detail, [07:40] but hopefully you can kind of see [07:42] the analogy between the middle and the top. [07:44] I'll have you talk about it later. [07:45] And then here's kind of the mind bender, [07:48] is we can extend the process to now involve [07:50] coordinated strand displacement by a set of these invader [07:53] strands that coordinate in a way to basically separate [07:57] the bottom block from the northeast block. [08:00] And in that way, we can have coordinated attack [08:02] by many invader strands and then split this very thick object [08:06] into two. [08:07] And then we can incorporate that into our crisscross growth [08:10] model where we're making these linear ribbons. [08:13] We introduce these coordinated toeholds [08:15] so you have a bunch of invader strands [08:16] that can eat in from the north and from the east. [08:20] And if you have invasion on two fronts, [08:22] then you can basically get this thing to split [08:24] in a stage of midnight animation showing this process. [08:28] Again, you can download it from the Google Doc [08:31] if you want to look at this in more detail. [08:34] And in that way, we can get exponential amplification. [08:39] And it kind of works, [08:41] but we're still trying to improve on its performance. [08:44] The latest is we have some kind of variant where we can get [08:46] down to subatomol or limited detection, [08:48] which we're very excited about. [08:49] So in summary, this is a scheme with [08:51] the single-stranded building blocks that we think will be [08:53] extremely useful for enzyme-free, [08:55] ultra-sensitive detection of things like pathogens, [08:58] especially in the developing world. [09:00] And I'll conclude by just reminding again [09:03] that here specifically we're looking at how do we [09:05] implement exponential conversion of [09:07] single-stranded building blocks into [09:09] double-stranded products for the purpose of diagnostics. [09:12] But on another scale, we're longitudinally interested, [09:14] how do we advance the field of [09:16] biomelecular nanotechnology to sustain [09:19] an exponential trend in the maximum complexity [09:22] of the structures that we can construct over time. [09:25] And then another thing I'd just like to point out is that [09:27] nothing in our crisscross cartoons [09:29] has anything to do with DNA. [09:30] We implemented this single-stranded DNA. [09:33] We implemented with DNA origami building blocks. [09:36] We challenge people in the community to [09:38] repeat this algorithm with other materials such as [09:40] proteins or maybe non-biological polymers as well. [09:43] So, thanks. [09:44] Interesting. Thank you. [09:48] Okay. We have time for one question. [09:54] Really, really simple question, [09:56] particularly related to the last point. [09:58] So, how does one of your deficit units compare to KT? [10:02] So, you had your energy deficit units, [10:06] how do they compare to KT? [10:08] Yeah. So, if you imagine, let's say your building blocks [10:10] were at one micromolar concentration, [10:13] and then the effective concentration within the ribbon, [10:16] let's just say for argument's sake it's one molar. [10:18] So, that's a six order of magnitude concentration to [10:22] pull it out of micromolar bulk solution into the ribbon. [10:26] So, each order of magnitude corresponds to 2.3 KT. [10:30] So, in the case of the origami, [10:33] there's 16 units, [10:36] 16 times 2.3 is like 38 KT. [10:39] That's huge. That's like two ATP's worth of energy. [10:42] It's never going to happen. It's not going to happen. [10:47] So, how do you think that [10:49] your work could fit into their respective architecture? [10:52] Yeah. So, I have this figure that I embedded in the work, [10:55] the workpiece and also it's here. [10:58] You can download it. [10:59] So, again, this is a scheme for making [11:02] an exploded view of pre-assembled 3-5 nanometer building blocks [11:07] that each would fit on the node on the crisscross grid. [11:11] Then the advantage here is that you could [11:14] hierarchically construct each slot with, [11:17] let's say, 32 guests on it. [11:18] So, first you have some kind of mechanism, [11:20] your first level printer makes [11:22] your 3-5 nanometer building blocks. [11:24] Then your second level process fabricates the slats [11:27] with 32 of them loaded up in a linear fashion. [11:30] This is the third step where we now assemble [11:34] those 32 subunits together into [11:36] a much bigger structure in an exploded view. [11:39] Then we can also do funny, [11:40] we have more access in this exploded view. [11:42] Then when we're ready, we could program it to [11:45] compact along the y-axis. [11:47] So, this is just like a little grid structure. [11:49] Hopefully, we've all seen these kind of [11:51] the boxing glove thing that expands and contracts. [11:53] So, contract in the y-axis. [11:56] Then what we do is we remove the DNA scaffold and we've [11:59] also inserted some rubber bands along the orthogonal axis. [12:04] So, in the second step, we can take the structure that's only [12:06] cross-linked along the y-axis and then we [12:10] can compact it along the x-axis as well. [12:13] It's probably confusing, but happy to talk about it more later. [12:16] Wonderful. Thank you so much, Will.