Functional DNA Nanotechnology: Precise Spatial and Dynamic Controls of Nanomaterials Morphology and Assembly and their Applications in Sensing, Imaging and Medicine
For general scope of this area, please see following review articles published in Acc Chem Res. (1), J. Mater. Chem. (2), and Curr. Opin. Chem. Biol. (3).
Figure 1. DNA “codes” for nanomaterials.
1. Discovery of DNA “Codes” for Nanomaterials Shapes and Morphologies
The shapes and morphologies of nanomaterials confer unique physical and chemical properties, making these nanomaterials excellent candidates for a wide range of applications, such as photonics, electronics, sensors and catalysis. It is well known that capping ligands play very important roles in influencing the shapes and morphologies of nanomaterials. As a result, previous studies have used small molecules, polymers, and peptide/proteins for this purpose (4). We have started a program of investigating the use of DNA as a programmable capping ligand that affords precise and systematic control of the length, charge, functional group, secondary and tertiary structures (5). Inspired by the discovery of the genetic code in biology, we have reported discovery of DNA codes for fine control of the shape and morphology of nanomaterials (6). Rules of shape control by difference DNA sequences and their combinations are summarized (Figure 1). These new DNA codes can play an important role in rational design and synthesis of novel nanomaterials with predictive shape control.
2. Time-dependent and Protein-directed In Situ Growth of Nanomaterials in Single Protein Crystals
Gold nanoparticles have been widely used in bionanotechnology, and yet the mechanism of biomolecule-directed nanoparticles formation remains to be elucidated. Similarly, while it has been established that biomolecules play a key role in the biomineralization of gold in biological species such as bacteria, the exact nature of the bio-nano interfacial interactions responsible for biomineralization is unknown. To address these issues, we have reported the use of intact, single lysozyme crystals to follow the time-dependent growth of gold nanoparticles, directed by the binding of Au(I) to lysozyme (Figure 2) (7).
Figure 2. Schematic views of gold nanoparticles formed within lysozyme crystals.
A protein crystal was employed instead of a protein solution because a crystal would not only slow down the fast kinetics of gold nanoparticle formation, allowing a careful mechanistic investigation, but also make the system amenable for structural characterization by high resolution X-ray crystallography. The time-dependent growth of gold nanoparticles growth and their three-dimensional distribution within the lysozyme crystals have been studied by STM together with electron tomography. Moreover, this approach did not disrupt the crystalline nature of the lysozyme crystals, thus allowing us to elucidate the reaction mechanism by X-ray crystallography. This study opens a new avenue for mechanistic studies of biomineralization and for synthesizing novel nanomaterial-in-protein crystal hybrid materials which may have applications in catalysis, optical and plasmonic devices, and sensing. Moreover, by applying this methodology to other inorganic systems, different nanoparticles have been successfully demonstrated to be formed inside protein crystals, creating new composite materials with optical and catalytic properties for potentially future applications (8).
Figure 3. Precise spatial control of nanomaterials and its extension to assembly of Janus nanoparticles.
3. Precise Spatial Controls of Nanomaterials Assembly
While a number of progresses have been made in synthesizing individual nanomaterials, the next level of challenge is how to assemble different nanomaterials together with precise spatial control under the same ambient conditions. DNA has been shown to be highly programmable molecule resulting in a number of 2D and 3D nanostructures. Despite the promise, functionalizing these structures is challenging. We have developed a novel method of using phosphorothioate DNA as anchors, and a bifunctional linker as a rigid molecular fastener that can connect nanoparticles to specific locations on the DNA backbone (9). Precise distance controls between two and three nanoparticles or proteins on double-stranded DNA with nanometer resolution have been demonstrated (Figure 3a).(10) This method has been further applied to 3D DNA structures by site-specific attachment of proteins onto a DNA tetrahedron through backbone-modified phosphorothioate DNA (11). Moreover, we have extended the DNA-based spatial control of nanomaterial assembly to asymmetric structures. In a step toward that goal, we demonstrated using DNA base pairing to form asymmetric nanostructures with spatial and chemical control on the platform of Janus nanoparticles (JNPs) (Figure 3b) (12).
Figure 4. Reversible nanoscale assembly.
4. Reversible Nanoscale Assembly
While much work has been devoted to control the pattern of nanoscale assembly, selectively reversible assembly of components in the nanoscale at specific sites has received much less attention. By taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we have demonstrated selective and reversible decoration of 2D DNA origami tiles with streptavidin, including revealing an encrypted Morse code “NANO” (Figure 4) (13). We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates.
Figure 5. Proof-reading and error removal in biology and in materials science.
5. How to make error-free or “perfect” nanomaterials
Despite tremendous progress made in self-assembly of nanomaterials, errors almost always occur in the assembly process. Even though rarely discussed in publications, those errors present a major obstacle for practical applications of nanomaterials in applications such as molecular electronics, photonics and computation. Inspired by nature, we have shown that a proof-reading unit can be designed to locate and remove error during or after the assembly process. Specifically, a DNAzyme can be designed to locate and remove errors in a DNA-templated gold nanoparticle assembly process (14). The concept is analogous to proof-reading and error removal in biology such as in protein synthesis process, and can be expanded to include many other biomolecules such as protein enzymes or biomimetic compounds such as chemical nucleases for controlling assembly of not only nanoparticles of defined particle sizes, shapes or compositions, but also other nanomaterials.
Figure 6. Stimuli-responsive assembly of different nanomaterials directed by functional DNA and their biosensing applications.
6. Dynamic Controls of Nanomaterials Assembly
In addition to precise spatial control, dynamic control of the assembly of nanomaterials in response to chemical stimuli under ambient conditions is also important. To meet this challenge, we took advantage of recent advance in biology to discover functional DNA, a new class of DNAs that can either bind to a target molecule (known as aptamers) or perform catalytic reactions (known as DNAzymes), that are very specific for a wide range of targets (1-2,15). We have demonstrated the use of these functional DNA for dynamic control of assembly of gold nanoparticles (14,16), iron oxide nanoparticles (17), quantum dots (18), and nanotubes (19), in response to a wide range of chemical and biological stimuli from small metal ions to large biomolecules, including cancer cell markers (20). Because these nanomaterials possess unique optical, electrical, magnetic and catalytic properties, these systems have been converted into colorimetric (21), fluorescent (22), electrochemical (19), photoluminescence sensors (23), and magnetic resonance imaging agents (24) for detection of a broad range of analytes with high sensitivity and selectivity (Figure 6).
Figure 7. A schematic view showing the targeting effects of nucleolin DNA aptamer-functionalized liposomes against MCF-7 cancer cells in vitro and in vivo.
7. Cell-Specific Imaging and Therapy with Aptamer-Functionalized Nanomaterials
The application of functional DNA nanotechnology has also been expanded to cell-specific imaging and therapy. Nucleic acid based aptamers provide excellent alternatives to antibodies as cell-specific agents and therefore are promising targeting ligands for targeted delivery systems (3,25). In addition, liposome is by far the most successful drug-delivery system and a number of liposome-based formulations have been approved by the US Food and Drug Administration for disease treatment in the clinic. We take the advantages of both systems and have designed the controlled formulation of aptamer-conjugated, anticancer drug-encapsulating multifunctional liposomes (Figure 7). Cancer-cell-specific targeting and drug delivery are demonstrated by using this delivery platform both in vitro and in vivo (26). Furthermore, we also show for the first time that a complementary DNA (cDNA) of the aptamer can function as an antidote to disrupt aptamer-mediated targeted drug delivery (26b). This strategy for reversible delivery can, in principle, be adapted to a broad range of chemotherapy agents. Moreover, we have further extended the aptamer-based targeting methods to other nanomaterials such as mesoporous silica (27) and upconversion nanoparticle (28), showing the excellent potential for cell-specific imaging, DNA delivery, and therapeutic applications.

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