# A logic-gated nanorobot for targeted transport of molecular payloads

Shawn M. Douglas*1, Ido Bachelet*1, George M. Church1

1 Wyss Institute for Biologically Inspired Engineering & Department of Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA

* These authors contributed equally to this work.

Correspondence to: George M. Church and requests for materials should be addressed to G.M.C. (gmc@harvard.edu, http://arep.med.harvard.edu/gmc/email.html).

## Abstract

We describe an autonomous DNA nanorobot capable of transporting molecular payloads to cells, sensing cell-surface inputs for conditional, triggered activation, and radical structural reconfiguration for payload delivery. The device can be loaded with a variety of materials in a highly organized fashion and is controlled by an aptamer-encoded logic gate, enabling it to respond to a wide array of cues. We implemented several different logical AND gates and demonstrate their efficacy in selective regulation of nanorobot function. As a proof of principle, nanorobots loaded with combinations of antibody fragments were used in two different types of cell-signaling stimulation. Our prototype could inspire new designs with different selectivities and biologically active payloads for cell-targeting tasks.

## Main Text

The DNA origami method1, in which a multiple-kilobase single-stranded 'scaffold' is folded into a custom shape by interaction with hundreds of oligonucleotide 'staple' strands, has proved to be extremely versatile for creating custom two- and three-dimensional assemblies2-4 that can template precise arrangement of diverse components5-7. DNA can also be used to construct devices that perform robotic tasks such as sensing, computation, and actuation8-11. Recently a three-dimensional DNA-origami box integrating both structural and computational components was described12. Inspired by these advances, we sought to design a robotic DNA device capable of selectively interfacing with cells to deliver signaling molecules to cell surfaces.

Using cadnano, a computer-aided design tool for DNA origami13, we created a nanorobot (or 'robot') in the form of a hexagonal barrel with dimensions of 35 x 35 x 45 nm3 in size. The barrel consists of two domains that are covalently attached in the rear by single-stranded scaffold hinges, and can be non-covalently fastened in the front by staples modified with DNA-aptamer-based locks (Fig. 1A). Initial self-assembly proceeds in a one-pot reaction in which 196 oligonucleotide staple strands direct a 7308-base M13mp18-derived scaffold strand into its target shape during a thermal-annealing ramp of rapid heating followed by slow cooling14.

A clasp system based on DNA locks and DNA keys was previously used to control the opening of the lid on a DNA box12. In order to operate our device in response to proteins, we designed a DNA-aptamer-based lock mechanism that opens in response to binding antigen keys (Fig. 1B,C). Our lock system was inspired by aptamer beacons15 and structure-switching aptamers16, which typically undergo target-induced switching between an aptamer-complement duplex and an aptamer-target complex. We incorporated aptamer-complement duplexes on the left and right sides of the front of the barrel such that the aptamer strands are attached to one domain and partially complementary strands are attached to the other domain. When both aptamers recognize their targets, the lock duplexes dissociate, and the nanorobot--acting as an entropic spring--undergoes a drastic reconfiguration to expose its previously sequestered surfaces. We found that shorter duplexes gave better sensitivity and faster activation rates, but at a cost of increased spontaneous activation. (See Note S2). We chose a lock duplex length of 23 bp for subsequent experiments because it displayed similar sensitivity to a shorter 16-bp duplex (activation at 10 pM [key]) without the unacceptable loss in sensitivity observed for longer duplexes (30, 37, and 44 bp required 1 nM [key]).

Payloads that were pre-modified by covalent attachment to the 5' end of a 15-base single-stranded DNA oligonucleotide linker were loaded inside the nanorobot. To enable multivalent interaction with surface receptors for potent cell stimulation17, we aimed to load at least two payload molecules per robot. To allow different payload orientations and spacings, 12 payload attachment sites are arranged in an inward-facing ring in the middle of the barrel (Fig. 1A,C). The sites are staple strands with 3' extensions that are complementary to a linker sequence attached to the intended cargo (Fig. 1D). After nanorobot folding, purification, cargo loading was carried out by adding linker-modified payload in molar excess to attachment sites and incubating at room temperature for 12 hours. Two types of cargo were loaded: 5-nm gold nanoparticles covalently attached to 5'-thiol-modified linkers18, and various Fab' antibody fragments that were covalently attached to 5'-amine modified linkers using a HyNic/4FB coupling kit (Solulink, San Diego, CA). We used negative-stain transmission electron microscopy (TEM) to analyze the device in closed and open states, with and without cargo (Fig. 1F). We observed by manual counting that on average four attachment sites were populated when loading gold nanoparticles and three sites were populated when loading antibody fragments (Fig. S11).

One obstacle to overcome in constructing a 'spring-loaded' device was to ensure assembly to high yield in its closed state. Like hands that set a mousetrap, we included two 'guide' staples incorporated adjacent to the lock sites that span the top and bottom domains of the device (Fig. 1E). The guide staples include 8-base toehold overhangs and could be removed after folding and purification steps by adding a 10:1 excess of fully complementary strands to the mixture19. We observed that folding with the aid of guide staples increased the yield of closed robots from 48% to 97.5% as assessed by manual counting of nanorobots images by TEM (Fig. S17).

To examine nanorobot function, a payload was chosen such that robot activation would be coupled to labeling of an activating cell (Fig. 2A). Robots loaded with fluorescently labeled antibody fragments against human HLA-A/B/C were mixed with different cell types expressing human HLA-A/B/C and various 'key' combinations (described below), and analyzed by flow cytometry. In the absence of the correct combination of keys, the robot remained inactive. In the inactive state, the sequestered antibody fragments were not able to bind the cell surface, resulting in a baseline fluorescence signal. However, when the robot encountered the proper combination of antigen keys, it was freed to open and bind to the cell surface via its antibody payload, causing an increase in fluorescence. We used key-neutralizing antibodies in competitive inhibition control experiments to verify that nanorobots are not activated by a non-ligand-based mechanism (Fig. S25).

The robot could be programmed to activate in response to a single type of key by using the same aptamer sequence in both lock sites. Alternatively, different aptamer sequences could be encoded in the locks to recognize two inputs. Both locks needed to be opened simultaneously to activate the robot. The robot remained inactive when only one of two locks is opened, regardless of which lock. The lock mechanism is thus equivalent to a logical AND gate, with possible inputs of cell-surface antigens not binding or binding ('0' or '1', respectively) to aptamer locks, and a possible outputs of remaining closed or a conformational rearrangement to expose the payload ('0' or '1', respectively) (Fig. 2B).

To test the generality and robustness of the aptamer-encoded logic gating, we designed six different robots using pairwise combinations of aptamer locks drawn from a set of three well-characterized aptamer sequences: antibody to platelet-derived growth factor20, shown in red; TE1721, shown in yellow; and sgc8c22, shown in blue (Fig. 2C). The six robot versions--plus a permanently-locked negative control and a no-lock positive control--were loaded with fluorescently labeled antibody to human HLA-A/B/C Fab' and used to probe six different cell lines, each expressing different profiles of the 'key' antigens recognized by the three chosen aptamers. A Burkitt's lymphoma cell line (Ramos) activated none of the robots. An acute myeloid leukemia cell line (Kasumi-1) activated all robots. A third cell line, isolated from a patient with large granular lymphocytic leukemia, aggressive NK type (NKL)23 activated robots with two antibody to PDGF locks, two TE17 locks, and with one antibody to PDGF and one TE17 lock. The baseline negative signal for NKL cells was higher than other cell lines because of NKL background fluorescence. A fourth cell line, isolated from acute T-cell leukemia (Jurkat), activated robots bearing two TE17 locks, two sgc8c locks, as well as one TE17 lock and one sgc8c lock. The fifth line was isolated from acute lymphoblastic leukemia (CCRF-CEM) and had a similar profile to Jurkat cells. Finally, a neuroblastoma cell line (SH-SY5Y) activated robots with two sgc8c locks. The lower intensity of the positive signals in the neuroblastoma cell line may be caused by the generally lower level of surface HLA-A/B/C expressed on these cells, and fluorescence intensities may be affected in general by expression levels of both aptamer keys and antibody payload targets.

We further examined the performance of the logic-gating mechanism in three ways. First, we tested the ability of the robot to selectively bind to a single cell type (NKLPTK7+/+) in a mixed population of two cell types (NKLPTK7+/+ and Ramos). Three versions of the robot were tested: a permanently closed version in which the guide staples were not removed, an open version in which no guide staples or aptamer locks were included, and a gated version using two sgc8c locks, which we expected to target only the PTK7-expressing NKL cells (Fig. 2D-F; Supporting Figs. S22-23). The gated version discriminated between the two cell types, only binding to NKL cells. Second, to further simulate physiological conditions, we mixed NKL cells with healthy whole-blood leukocytes and found that robots gated with antibody to PDGF locks discriminated those NKL cells with high precision (Fig. 2G; Supporting Fig. S19). Third, we examined the sensitivity of the nanorobots to cell concentration using a background of 1e6 Ramos cells, tested for the ability of an sgc8c-gated robot to bind to JurkatPTK7+/+ cells. Nanorobots were activated for every cell count, from 1e6 cells down to the single-cell level (Fig. S24).

Finally, we investigated the ability of an activated robot to interface with cells and stimulate their signaling in separate inhibition and activation tasks. We first chose to target NKL cells using a pair of anti-PDGF locks, and loaded the robots with a combination of anti-human CD33 and antibody to human CDw328/Siglec-7 Fab' fragments, which have been shown to induce growth arrest in leukemic cells24. The robots induced growth arrest in NKL cells in a dose-dependent fashion (Fig. 3A,B). The robots suppressed Jun N-terminal Kinase (JNK) (Fig. 3C) and Akt/protein kinase B signaling in this process (Fig. S27). We then used T cells to test various methods of signaling-pathway activation (Fig. 3D-F). Robots loaded with combination of antibody to human CD3e Fab' and antibody to flagellin Fab' were mixed with T cells and found to induce activation (Fig. 3D,F). Previous work has shown that flagellin can augment T-cell activation25. We found that our nanorobots were able to collect flagellin from a 100 pg/mL solution and induce augmented T cell activation (Fig. 3E,F). These findings demonstrate that the robots can induce a variety of tunable changes in cell behavior. Furthermore, biologically active payloads may be bound indirectly via interactions with antibody fragments, enabling applications in which the robot carries out a scavenging task prior to targeted payload delivery.

## Contributions

S.M.D. and I.B. designed and performed the experiments and analyzed the data. S.M.D., I.B., and G.M.C wrote the manuscript.

## Acknowledgments

We thank J. Ritz for providing NKL cells; C. Reynolds for technical assistance with NKL cells; C. Strong and G. McGill for assistance with 3D visualization using Molecular Maya; A. Marblestone for helpful discussions; and J. Markson for comments on the manuscript. S.M.D. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund and a Wyss Technology Development Fellowship, and I.B. is supported by a fellowship from the Life Sciences Research Foundation.

## Figure Captions

### Fig. 1
Design and TEM analysis of aptamer-gated DNA nanorobot. (A) Schematic front orthographic view of closed nanorobot loaded with a protein payload. Two DNA-aptamer locks fasten the front of the device on the left (boxed) and right. (B) Aptamer lock mechanism, consisting of a DNA aptamer (blue) and a partially complementary strand (orange). The lock can be stabilized in a dissociated state by its antigen key (red). Unless otherwise noted, the lock duplex length is 24 base pairs, with an 18-24 base thymine spacer in the non-aptamer strand. (C) Perspective view of nanorobot opened by protein displacement of aptamer locks. The two domains (blue and orange) are constrained in the rear by scaffold hinges. (D) Payloads such as gold nanoparticles (gold) and antibody Fab' fragments (magenta) can be loaded inside the nanorobot. (E) Front and side views show guide staples (red) bearing 8-base toeholds aid assembly of nanorobot to 97.5% yield in closed state as assessed by manual counting. After folding, guide staples are removed by addition of fully complementary oligos (black). Nanorobots can be subsequently activated by interaction with antigen keys (red). (F) TEM images of robots in closed and open conformations. Left column: unloaded, center column: robots loaded with 5 nm gold-nanoparticles, right column: loaded with Fab' fragments. Scale bars: 20 nm.

### Fig. 2
Analysis of logic-gated nanorobot activation by different cell types. (A) Experimental scheme. Nanorobots were loaded with fluorescently labeled antibody Fab' fragments to human HLA-A/B/C. In their unlocked state, nanorobots will bind to any cell expressing the HLA-A/B/C antigen (top). They remain inactive in the presence of key(-) cells (middle), but activate upon engaging key(+) cells (bottom). (B) Truth table for nanorobot activation. The aptamer-encoded locks function as an AND gate responding to molecular inputs (keys) expressed by cells. Color match signifies lock and key match. Aptamer-antigen activation state serves as input, while output is manifested as nanorobot conformation. (C) Eight different nanorobot versions (10-100 fmol, loaded with anti-HLA-A/B/C antibody fragments at a molar excess of 20) were tested with six different cell types expressing various combinations of antigen keys by incubating for 5 h. Each histogram displays the count of cells versus fluorescence due to anti-HLA-A/B/C labeling. (D) NKL cells (50,000/sample, with transiently induced expression of PTK7 (NKLPTK7+/+), Ramos cells (100,000/sample), FITC-anti-human-CD20 (0.1 ug/mL), and permanently locked robots loaded with APC-anti-human HLA A/B/C Fab' were incubated at room temperature for 5 h. No labeling was observed, as locked robots remain inactive. (E) Unlocked robots react with both cell populations. (F) sgc8c-gated robots react only with the cell population expressing the PTK7 key. (G) Forward- vs. side-scatter dot plot of 4:1 mixture healthy human whole-blood leukocytes and NKL cells. Nanorobots loaded with anti-CD33 Fab' payload and gated with anti-PDGF locks selectively label NKL cells. Off-target binding occurred in 0.6% of sampled cells.

### Fig. 3
Nanorobots manipulate target cell signaling. (A) Experimental scheme. A single dose of nanorobots loaded with an equal mixture of anti-human CD33 and anti-human CDw328/Siglec-7 Fab' fragments (cyan, magenta, respectively, each at a molar excess of 10 over nanorobots) and gated by anti-PDGF locks (blue lines) were used to treat NKL cells at various concentrations (0-100 nM). Phosphorylation of JNK was measured after 72h by intracellular flow cytometry. (B) NKL cells (10,000/sample) treated with nanorobots were analyzed after 72h for cell cycle distribution by propidium iodide (5 ug/mL). (C) Phosphorylation level of JNK as a function of robot concentration (lowest, 0; highest, 100 nM) after 72 h. Error bars for B and C represent SEM of 2 biological replicates. (D-E) Incremental activation of T cells by nanorobots loaded with anti-human CD3e (blue) and anti-flagellin Fab' (black). Nanorobots (50 nM) with (E) and without (D) pre-incubation with flagellin (red) (100 pg/mL) were reacted with Jurkat T cells (pink) for 1 h at 37C. (F) Histograms showing T-cell activation following nanorobot treatment, as measured by labeling with FITC-anti-CD69.

## References and Notes

1. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297-302, doi:10.1038/nature04586 (2006).
2. Ke, Y. et al. Scaffolded DNA origami of a DNA tetrahedron molecular container. Nano Lett 9, 2445-2447, doi:10.1021/nl901165f (2009).
3. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414-418, doi:10.1038/nature08016 (2009).
4. Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725-730, doi:10.1126/science.1174251 (2009).
5. Kuzuya, A. et al. Precisely programmed and robust 2D streptavidin nanoarrays by using periodical nanometer-scale wells embedded in DNA origami assembly. Chembiochem 10, 1811-1815, doi:10.1002/cbic.200900229 (2009).
6. Voigt, N. V. et al. Single-molecule chemical reactions on DNA origami. Nat Nanotechnol 5, 200-203, doi:10.1038/nnano.2010.5 (2010).
7. Stephanopoulos, N. et al. Immobilization and one-dimensional arrangement of virus capsids with nanoscale precision using DNA origami. Nano Lett 10, 2714-2720, doi:10.1021/nl1018468 (2010).
8. Stojanovic, M. N. & Stefanovic, D. A deoxyribozyme-based molecular automaton. Nature Biotechnology 21, 1069-1074, doi:10.1038/nbt862 (2003).
9. Seelig, G., Soloveichik, D., Zhang, D. Y. & Winfree, E. Enzyme-Free Nucleic Acid Logic Circuits. Science 314, 1585-1588, doi:10.1126/science.1132493 (2006).
10. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro, E. An autonomous molecular computer for logical control of gene expression. Nature 429, 423-429, doi:10.1038/nature02551 (2004).
11. Ding, B. & Seeman, N. C. Operation of a DNA robot arm inserted into a 2D DNA crystalline substrate. Science 314, 1583-1585, doi:10.1126/science.1131372 (2006).
12. Andersen, E. S. et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 459, 73-76, doi:10.1038/nature07971 (2009).
13. Douglas, S. M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res 37, 5001-5006, doi:10.1093/nar/gkp436 (2009).
14. Design blueprints, preparation methods, and DNA sequences are available the Supporting Online Material, Figures S1-S4, and Table S1.
15. Hamaguchi, N., Ellington, A. & Stanton, M. Aptamer beacons for the direct detection of proteins. Anal Biochem 294, 126-131, doi:10.1006/abio.2001.5169 (2001).
16. Nutiu, R. & Li, Y. Structure-switching signaling aptamers. J Am Chem Soc 125, 4771-4778, doi:10.1021/ja028962o (2003).
17. Boniface, J. J. et al. Initiation of Signal Transduction through the T Cell Receptor Requires the Multivalent Engagement of Peptide/MHC Ligands. Immunity 9, 459-466, doi:10.1016/s1074-7613(00)80629-9 (1998).
18. Alivisatos, A. P. et al. Organization of 'nanocrystal molecules' using DNA. Nature 382, 609-611, doi:10.1038/382609a0 (1996).
19. Yurke, B., Turberfield, A. J., Mills, A. P., Jr., Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605-608, doi:10.1038/35020524 (2000).
20. Green, L. S. et al. Inhibitory DNA ligands to platelet-derived growth factor B-chain. Biochemistry 35, 14413-14424, doi:10.1021/bi961544+ (1996).
21. Tang, Z. et al. Selection of Aptamers for Molecular Recognition and Characterization of Cancer Cells. Analytical Chemistry 79, 4900-4907, doi:10.1021/ac070189y (2007).
22. Huang, Y. F. et al. Molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells. Chembiochem 10, 862-868, doi:10.1002/cbic.200800805 (2009).
23. Robertson, M. J. et al. Characterization of a cell line, NKL, derived from an aggressive human natural killer cell leukemia. Exp Hematol 24, 406-415 (1996).
24. Vitale, C. et al. Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: engagement of CD33 induces apoptosis of leukemic cells. Proc Natl Acad Sci U S A 98, 5764-5769, doi:10.1073/pnas.091097198 (2001).
25. Ye, Z., Lee, C. M. L., Sun, G. W. & Gan, Y. H. Burkholderia pseudomallei Infection of T Cells Leads to T-Cell Costimulation Partially Provided by Flagellin. Infection and Immunity 76, 2541-2550, doi:10.1128/iai.01310-07 (2008).