Notes on sequence programmability in bio-templated electronics

Note: prepared as a response to this RFI.

Response to prompt (b) on Biotemplated Registration capabilities:

b1. What are the physical mechanisms underlying your registration approach(es)?  Include surface chemistry requirements in your discussion.

The proposed approach would achieve sequence-specific addressable chips that can direct unique-sequence DNA origami to specific spots on chip with an exponential diversity of sequence programmability rather than a more limited diversity of shape and surface affinity programmability as in previous work. 

To do this, one needs to be able to approximately size-match single DNA origami-like structures with single sequence-specific spots on chip (think of them as localized “forests” of copies of a particular DNA sequence on chip). One way of doing that would be to photo-pattern a sequence-specific DNA microarray, and then shrink the spots with Implosion Fabrication. 

What are the physical principles underlying Implosion Fabrication? It turns out that there are materials called hydrogels that, when you put them in water, can swell uniformly by a large factor, say 10x along each axis. If you add salt, they uniformly shrink back down. Implosion Fabrication uses a focused spot of light to pattern materials into a swollen hydrogel, and then shrink. So you can get 10x better resolution, say, then the smallest diameter of a focused spot of light, i.e., you can get 10’s of nanometer resolution using light with wavelength of hundreds of nanometers. This can be done with a variety of materials, but here is shown just with fluorescent dyes for demonstration purposes:

A key advantage of this approach is that Implosion Fabrication operates directly in three dimensions. 

b2. Does your approach incorporate biomaterials into the resulting device?

It can, in theory, though this depends on the nature of the post-processing, e.g., whether it involves high temperatures. The approach could be adapted for different such scenarios.  

b3. What are the expected capabilities of your registration approach(es) (e.g., location, orientation and geometrical tolerances, pitch, critical dimensions, error in critical dimensions, density multiplication, critical dimension shrinkage)?  Please include a discussion of how computational and metrology resources assist in this approach.

Goal: The key goal would be to take the full addressability within DNA origami — the fact that each staple strand, which goes to a unique site on the origami, with few nanometer precision, say, and can thus bring a unique attached chemical, nanoparticle or so on to that particular site on the origami (the 2007 Battelle roadmap has a good description of this concept, they call it “unique addressing”) — and extend that to an area approaching the size of a computer chip, so say a millimeter on a side instead of 100 nm on a side.

Background: What the current state of the art can do is use electron beam lithography to make small “sticky” spots (I’m glossing over the chemistry obviously) on a silicon surface — and importantly, those spots can have a well defined orientation and be of the exact right size and shape to stick to a shape-matched DNA origami. Like this: note how the DNA origami triangles line up quite well inside the lithographic triangles:

They can then use this to make some basic photonic devices. This is one of those technologies where it feels like it now needs exploration to find its killer app. One possibility is positioning of a small number of discrete photonic components at the right locations on chips, e.g., for single photon sources — there is some progress in that general direction: “the authors were able to position and orient a molecular dipole within the resonant mode of an optical cavity”.

Proposed innovations: The proposed approach would go beyond just matching the shapes of lithographic spots to the shapes of DNA origami, and instead to actually have unique DNA sequences at unique spots that could uniquely bind to a given DNA origami. This would be a combination of a few technologies

In more detail, in my mostly theoretical thesis chapter on “nm2cm” fabrication

http://web.mit.edu/amarbles/www/docs/Marblestone_nm2cm_thesis_excerpt.pdf

http://web.mit.edu/amarbles/www/docs/Bigger-NanoBots-Marblestone.pdf

https://dash.harvard.edu/handle/1/12274513

we proposed that the key gap in this field — of integrating biomolecular self-assembly with top-down nanofabrication to construct chip-scale systems — is that, as impressive are works like Ashwin Gopinath’s using shape to direct DNA origami to particular spots on chips, it would be even more powerful if we could direct specific origami to specific spots on chip in a sequence-specific way: each spot on the chip should have a unique DNA address that could match to a unique DNA origami slated to land there. How can we do that? 

1.1) Optical approaches (faster, cheaper than electron beam lithography) can deposit or synthesize particular DNA sequences at particular spots on chip — and this is widely used to create DNA microarrays — but the spot sizes and spacings of the resulting DNA “forests” are too large to achieve “one origami per spot”

http://www.biostat.jhsph.edu/~iruczins/snp/extra/05.08.31/nbt1099_974.pdf

1.2) So we proposed to combine sequence non-specific but higher resolution photolithography to make small spots, with coarser grained optical patterning to define sequences for those spots, and then large origami rods spanning spot to spot to help ratchet orientation and spacing into a global “crystal-like” pattern: see nm2cm chapter above

Anyway, we didn’t demonstrate much of this experimentally at all (alas, it needed an ARPA program not just a rather clumsy grad student, or at least that would be my excuse!), but since then

1.a) Implosion Fabrication (ImpFab), mentioned above, may now provide a way to take a sequence-specific DNA microarray and shrink it so that the spot size matches achievable sizes of DNA origami. Something like this: note the tiny DNA origami in the lower right for scale

1.b) Researchers have started making smaller/finer-resolution microarray-like sequence-specific (albeit random) patterns on chips, and even transferred them to other substrates

https://www.biorxiv.org/content/10.1101/2021.01.25.427807v1.full

https://www.biorxiv.org/content/10.1101/2021.01.17.427004v1.full

(With these, you make a fine grained but random pattern and then image/sequence it in-situ to back out what is where. This would obviously entail a significant metrology component, i.e., figuring out what sequence is where and then synthesizing a library of adaptor strands to bring the right origami to the right sequences.)

1.c) DNA origami have gotten bigger, too, closer to matching the sizes even of existing non-shrunken microarray spots

https://www.nature.com/articles/nature24655

1.d) Another approach that could be used for fine-grained, sequence specific patterning would be something like ACTION-PAINT. This is in the general category of nanopatterning via “running a microscope in reverse”. Basically, there is a microscopy method called DNA PAINT that works like this. You have some DNA strands on a surface, arranged just nanometers apart from one another, and you want to see how they are all arranged. If you just put fluorescent labels on all of them at once, and look in an optical microscope, then the limited resolution of the optical microscope — set by the wavelength of light, a few hundred nanometers — blurs out your image. But if you can have complementary DNA strands bind and unbind transiently with the strands on the surface, fluorescing only when they bind, and such that at any given time only one is bound, then you can localize each binding event, one at a time, with higher precision than the wavelength (by finding the centroid of a single Gaussian spot at a time). That’s the basic principle of single-molecule localization microscopy, which won a Nobel Prize in 2018

The magic is that you can localize the centroid of one (and only one) isolated fluorescent spot much more precisely than you can discriminate the distance between two (or more) overlapping fluorescent spots. So you rely on having a sparse image at any one time, as DNA molecules bind on and off to different sites on the object such that typically only one site has a bound partner at any given time on, and then you localize each binding event one by one and build up the overall image as a composite of those localizations.

Anyway, that’s a microscopy method that lets you see with resolution down to a couple nanometers, well below the wavelength of light.

But how can you use this for nano-patterning? Well, imagine you have a desired pattern you want to make, and you are doing this “single molecule localization microscopy” process in real time. Then, if you can detect that a DNA strand has bound to a spot that is supposed to be part of your pattern, and you can register this in real time, then you can quickly blast the sample with a burst of UV light which locks that strand in place, preventing it from ever leaving again. That “locks in” a DNA bound to that spot. Now, most of the time, the localizations you’ll see will be at spots you don’t want to be part of your pattern, so you don’t blast the UV light then. But every so often, you’ll see a probe bound at a spot you want to be in the pattern, and when that happens, you take fast action, locking it in. That’s what ACTION-PAINT does:

This can be seen as a kind of molecular printer with in principle roughly the same resolution as that of the underlying single molecular localization microscopy method. Which in practice is not quite as high as the best AFM positioning resolution. But it is pretty high, in the single digit nanometers in the very best case. 

Thus, I think sequence-specific bio-chips, in which thousands of distinct origami as defined by sequences, not just a few as defined by shapes, can be directed to their appropriate spots on chip in a multiplexed fashion, should be possible. Exactly what their killer applications would be is less clear to me as of now.  

b4. How broadly can your approach be applied (i.e., is it limited to a single material and/or device)?

The approach would constitute a general platform for 3D hierarchical multi-material nanofabrication. If developed intensively, many thousands of different DNA origami bearing different functionalizations could in theory be brought to appropriate defined locations in 3D. Orientation of parts would be challenging to achieve but see the “nm2cm” crystal-like annealing process proposed below to above to allow this. Other materials could also be patterned in-situ using the standard implosion fabrication methods. 

b5. What constitutes a defect in your approach?

One could have a) defective origami, b) spots that are not patterned with DNA, c) spots with DNA that do not receive the right origami, d) other larger-scale defects, e.g., non-uniformities in the implosion process if using implosion fabrication, e) orientation defects if aiming to achieve defined orientations, e.g., in something like the nm2cm scheme.

b6. What defect rate and/or density can your approach achieve?

Currently unknown. In theory, layers of error correction could be applied at various levels to reduce defect rates. 

b7. Can defect reduction techniques be applied to your approach and if so, what is the expected impact?

Exact design scheme and quantitative impact not yet clear.

b8. What manufacturing throughput can your approach achieve?

Because it can rely on photolithography rather than electron-beam lithography, and pattern on the origami at the few-nm scale in a massively parallel way, the approach could potentially be very fast, e.g., with holography optical patterning of the initial template to be imploded. 

In general, for all detailed implementation questions here, it should be noted this is more of a set of design concepts and these are quite early-stage. In my mind this would form an ancillary, more speculative part of a program, aiming to seed sequence specific assembly and registration principles beyond the “bread and butter” parts of a program that might involve aspects closer to the published literature on registration in 2D by, for example, Gopinath/Rothemund et al.

b9. What existing nanomanufacturing infrastructure (e.g., tooling, processes) is required to enable your approach?  Are these resources currently available to you?

I am currently doing other kinds of work more on the institutional side. Would suggest doing this via groups like Irradiant Technologies and collaborations with DNA nanotechnology (e.g., Ashwin Gopinanth, William Shih) and DNA microarray fabrication (e.g., Franco Cerrina, Church lab, spatial transcriptomics labs using related methods) groups. In other words, I’m not in a direct position to execute on this experimentally right now.

b10. What computational resources would assist in simulating your approach?  If you could design the ideal computational infrastructure/ecosystem, what would it look like?  Please be quantitative with expected gains from having access to this ecosystem.

Depends on further narrowing down what this gets used for. Computing doesn’t seem to be the key limitation right now for this project. 

b11. In what way(s) are these resources different from what is currently available?

Computing doesn’t seem to be the key limitation right now for this project. 

b12. How and to what magnitude would these computational resources assist your approach (e.g., improving throughput, decreasing defects, predicting device characteristics)?

Computing doesn’t seem to be the key limitation right now for this project. 

b13. What are the expected resource requirements for your approach (e.g., raw materials required, power, water)? 

Comparable to DNA microarray manufacturing. 

b14. What are the expected costs (including waste streams) of your approach and how do they compare to existing approaches?

Comparable to DNA microarray manufacturing. 

b15. What metrology tools are needed to achieve the capabilities of your registration approach?  If you could design the ideal infrastructure/ecosystem, what would it look like? 

Depending on whether one does random patterning of the initial sequences and then reads them out, this may need something like an Illumina sequencing machine to read the locations of the sequences prior to implosion and addition of the DNA origami. 

b16. In what way(s) are these metrology resources different from what is currently available?

Just needs adaptation of detailed protocols. 

References

Oran D, Rodriques SG, Gao R, Asano S, Skylar-Scott MA, Chen F, Tillberg PW, Marblestone AH, Boyden ES. 3D nanofabrication by volumetric deposition and controlled shrinkage of patterned scaffolds. Science. 2018 Dec 14;362(6420):1281-5.

Marblestone AH. Designing Scalable Biological Interfaces (Doctoral dissertation, Harvard, 2014).

Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nature biotechnology. 1999 Oct;17(10):974-8.

Cho CS, Xi J, Park SR, Hsu JE, Kim M, Jun G, Kang HM, Lee JH. Seq-Scope: Submicrometer-resolution spatial transcriptomics for single cell and subcellular studies. bioRxiv. 2021 Jan 1.

Chen A, Liao S, Ma K, Wu L, Lai Y, Yang J, Li W, Xu J, Hao S, Chen X, Liu X. Large field of view-spatially resolved transcriptomics at nanoscale resolution. bioRxiv. 2021 Jan 1.

Liu N, Dai M, Saka SK, Yin P. Super-resolution labelling with Action-PAINT. Nature chemistry. 2019 Nov;11(11):1001-8.

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