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Nanorobots and Devlivery of Pharmaceutics
Nanorobots and Devlivery of Pharmaceutics
consists of two halves connected by a switchable hinge (Fig. 1). Two distinct DNA aptamers are used to close and lock the DNA barrel. Because each of the aptamers specifically binds different protein antigens, they form an AND logic gate that requires the presence of two protein targets to be activated. When both aptamers bind their targets, a conformational change in the barrel releases the cargo. Thus, the aptamer-encoded lock functions as a sense-compute-actuate mechanism that could in principle be deployed to trigger a specific therapeutic response. In a first set of experiments, Douglas etal.1 used their aptamer-gated DNA nanobots to detect selected biomarkers (such as plateletderived growth factor or protein tyrosine kinase 7) expressed on the surface of leukemia cells. Several combinations of three aptamers that recognize different surface antigens were incorporated into the nanobot. To characterize the system, the authors used fluorescently labeled Fab antibody fragments as the payload; the nanobots showed highly specific binding to the cells that displayed the correct combination of surface antigens, even in mixed populations of whole-blood leukocytes. Next, the nanobots were used to activate signaling pathways in target cells. The authors loaded the nanobots with Fab antibody fragments known to bind human CD33 and human CDw328 and induce growth arrest in leukemic cells. Upon recognition of the surface antigen PDGF on cells from a patient with aggressive lymphocytic NK-type leukemia, the barrel structure opened, allowing the antibody payload
to bind to cell-surface receptors and inhibit the growth of the target cells. Similarly, an increase in T cell activation was induced by a nanobot loaded with Fab fragments specific for human CD3 and flagellin. Additional measurements that quantify the AND gates sensitivity to the inputs, the rate of erroneous nanobot activation and the correlation between the number of activated nanobots with successfully modified cell signaling pathways would be required to fully evaluate nanobot performance. The aim of smart drug-delivery systems is to administer smaller drug doses to patients while offering improved therapeutic efficiency and fewer side effects compared with conventional drug delivery methods. Notable recent developments have included systems based on liposomes, polymersomes, micelles, nanoparticles and antibodies9. Yet these approaches do not provide the wide range of design modularity and structural programmability that DNA nanotechnology affords. Much work remains before the approach of Douglas et al.1 could be used for drug delivery in vivo. For example, nanobots might not be stable in the presence of nucleases and other enzymes in the blood. Increased resistance to degradation may be achievable by methods such as chemical cross-linking of selected DNA strands or the use of peptide nucleic acids or locked nucleic acids. Similarly, aptamerencoded locks may lose specificity and efficiency in protein-rich serum. In addition, delivery into cells is required for broad application of a drug-delivery system
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Hexagonal barrel
Aptamer lock
Origami self-assembly
Payload
DNA nanorobot
Figure 1 DNA nanobot for targeted drug delivery. To fabricate the nanobot by DNA origami, a long single-stranded DNA scaffold is folded into the shape of a hexagonal barrel by short oligonucleotide staple strands. The assembled nanobot consists of two aptamer-encoded locks, a barrel-shaped body and a bound molecular cargo. The use of two different aptamers that unlock when exposed to two antigens results in an AND logic gate, meaning that the nanobot opens in the presence of the correct combination of antigen keys. The molecular payload is then released to bind to target cells and activate signaling pathways.
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Katie Vicari
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given the many potential intracellular targets, and large DNA nanostructures are believed to have difficulty penetrating biological membranes. The display of amphiphilic molecules or cell-penetrating peptides on the surface of nanobots might facilitate tissue penetration and cellular uptake. Another concern is that DNA-origami containers, which can require nearly 200 unique oligonucleotides, are structurally more complicated than liposomes and many other drug carriers. For large-scale production, it may be necessary to minimize the number of constituent strands and reduce the complexity of the design, perhaps to even a single DNA strand10. Despite these challenges, it is conceivable that DNA nanobots could one day be used to influence the gene expression or metabolic pathways of target cells11. As with any drug-delivery strategy, improved understanding
2012 Nature America, Inc. All rights reserved.
of systems biology in health and disease will be crucial for the ultimate success of this approach.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
1. Douglas, S.M., Bachelet, I. & Church, G.M. Science 335, 831834 (2012). 2. Pinheiro, A.V., Han, D., Shih, W.M. & Yan, H. Nat. Nanotechnol. 6, 763772 (2011). 3. Cho, E.J., Lee, J.W. & Ellington, A.D. Annu. Rev. Anal. Chem. 2, 241264 (2009). 4. Qian, L. & Winfree, E. Science 332, 11961201 (2011). 5. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro, E. Nature 429, 423429 (2004). 6. Rothemund, P.W.K. Nature 440, 297302 (2006). 7. Lund, K. et al. Nature 465, 206210 (2010). 8. Andersen, E.S. et al. Nature 459, 7376 (2009). 9. Lammers, T., Aime, S., Hennink, W.E., Storm, S. & Kiessling, F. Acc. Chem. Res. 44, 10291038 (2011). 10. Shih, W.M., Quispe, J.D. & Joyce, G.F. Nature 427, 618621 (2004). 11. Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye, F.A. Science 333, 470474 (2011).
In previous work, single-cell analysis has been used to define the clonal architecture of acute lymphoblastic leukemia5 by multiplex fluorescence in situ hybridization and to define the tumor evolution of breast cancers6 using next-generation sequencing to quantify genomic copy number within individual nuclei. In both cases, the common progenitor clone of the diverse subclones was marked and identified by genome aberrations (chromosomal translocations and/or copy-number alterations). The outstanding question was whether somatic variants would reveal a similar clonal pattern at the sequence level. Understanding this issue in detail will have profound clinical implications. There are now several targeted therapies directed against cancer genes that are mutated (such as BRAF and EGFR) or amplified (such as HER2), which some have called actionable mutations. Tumor heterogeneity poses a considerable challenge to such therapies. For example, how will clinicians decide whether a targeted therapy is indicated if the mutation is present in only a minor proportion of cancer cells? Hou et al.2 developed a novel method for genome sequencing of single cells. First, they
isolated the cells using an inverted microscope and microcapillary pipetting followed by whole-genome amplification (WGA) based on multiple-displacement amplification with the 29 enzyme, which allowed linear amplification of the complete genome. WGA DNA was then analyzed by massively parallel sequencing with Illumina instruments. The authors developed and validated the method with two single cells from a lymphoblastoid cell line isolated from the individual whose DNA was used for the first Asian diploid genome sequenced. This work showed that whole-genome sequencing of WGA DNA has acceptable error rates, and it provided a baseline for subsequent studies of single cells from cancer samples using only exome sequencing. Next, Hou et al.2 sequenced the exomes of 58 single cancer cells from an individual with essential thrombocythemia, a myeloprolifera tive neoplasm, to a mean depth of 30. Using mutation-calling algorithms, which were experimentally validated both by comparing all single-cell somatic variants to those identified in whole essential-thrombocythemia tissue sequencing and by PCR-Sanger sequencing of 30 randomly selected mutations, the authors identified 712 somatic variants, of which 171 coding variants were further assessed. Seventy-eight of these 171 coding somatic mutations (in a total of 71 genes) were nonsynonymous. A population-genetics analysis of these data revealed the monoclonal origin of essential-thrombocythemia cells in this patient. Furthermore, a driver-gene prediction model identified eight genes as the ones with the highest likelihood of being involved in the neoplastic initiation and/or progression of essential thrombocythemia. In a second study from the same laboratory, Xu et al.3 applied the single-cell sequencing method to analyze the exomes of 25 single cells from a renal cell carcinoma20 from the tumor and 5 from adjacent normal tissue. The sequencing data revealed 260 coding somatic mutations. Principal component analysis showed that three of the single cancer cells clustered tightly with the five normal single cells, which the authors took to indicate that these were actually healthy cells admixed within the tumor. The remaining 17 single cancer cells had 229 somatic mutations, and these mutations were used to determine the allelic frequency at the 229 nucleotide positions. This analysis revealed two distinct peaks, one with a frequency range of 05% and the other with a frequency range of 1520%. Thus, not only was there significant intratumoral heterogeneity, but alsonotablythere were no dominant clones identified in the cancer tissue. In other words,
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