Spherical nucleic acid

Spherical nucleic acids (SNAs)[1] – defined as structures that are an arrangement of densely packed, highly oriented nucleic acids in a spherical geometry – were first introduced in 1996[2] by the Mirkin group at Northwestern University. The arrangement and orientation of one-dimensional linear nucleic acids within this three-dimensional framework results in new chemical, biological, and physical properties in the use of nucleic acids for intracellular gene regulation (though this is disputed [3]), molecular diagnostics, and materials synthesis applications.

Figure 1. Three important classes of nucleic acids: one-dimensional linear, two-dimensional circular, and three-dimensional spherical.[1]

Structure and Function

The first SNA consisted of 3’ alkanethiol-terminated, single-stranded oligonucleotides (short DNA sequences) covalently attached to the surface of spherical gold nanoparticles. A dense nucleic acid loading was achieved through a series of salt additions, in which positively charged counterions were used to reduce electrostatic repulsion between adjacent negatively charged DNA strands and thereby enable more efficient DNA packing onto the nanoparticle surface.

One- and two-dimensional forms of nucleic acids (e.g., single strands, linear duplexes, and plasmids) are important biological machinery for the storage and transmission of genetic information (Fig. 1). Underlying this utility are the bioprogrammable interactions between complementary nucleotide bases. Scientists and engineers have been synthesizing and, in certain cases, mass-producing such structures for decades in an effort to understand and exploit this elegant recognition motif. The recognition abilities of nucleic acids are enhanced when arranged in a spherical geometry, which allows for polyvalent interactions. This polyvalency, along with the high density and degree of orientation described above, helps to explain why a three-dimensional nucleic acid structure composed of linear, one-dimensional nucleic acids exhibits different properties than its lower-dimensional constituents (Fig. 2).

Figure 2. Properties of spherical nucleic acids (SNAs) versus linear nucleic acids.[1]

Two decades of research on SNAs have revealed that they combine properties from the inorganic core and the nucleic acid shell. The inorganic nanoparticle core (in addition to gold, silver,[4] iron oxide,[5] silica,[6] and semiconductor materials[7] have also been used) serves two purposes: 1) it imparts upon the conjugate novel physical and chemical properties (e.g. plasmonic, catalytic,[8][9] magnetic,[5] luminescent[7]), and 2) it acts as a scaffold for the assembly and orientation of nucleic acids. The nucleic acid shell gives the conjugate chemical and biological recognition abilities that offer greater binding strengths and higher duplex stabilities compared to the same sequence of linear DNA,[8][10][11] cooperative melting behavior with DNA strands of a complementary sequence, and enhanced cellular uptake without the use of transfection agents.[12] Recent studies have shown that one can crosslink the DNA strands at their base, and subsequently dissolve the inorganic core with KCN or I2 to create a coreless form of SNA (Fig. 3, right),[13] which exhibits many of the properties of the original DNA-nanoparticle conjugate (Fig. 3, left).

Figure 3. Gold nanoparticle filled and core-less spherical nucleic acid structures (SNAs).[1]

Due to their structure and function, SNAs occupy a materials space distinct from "DNA nanotechnology and origami."[14][15][16] With DNA origami, such structures are synthesized via DNA hybridization events. In contrast, the SNA structure can be synthesized independent of nucleic acid sequence and hybridization, instead relying upon chemical bond formation between nanoparticles and nucleic acid adsorbates. Furthermore, DNA origami uses DNA hybridization interactions to realize a final structure, whereas SNAs and other forms of three-dimensional nucleic acids (structures templated with a triangular prism, rod, octahedra, or rhombic dodecadhedra-shaped nanoparticles)[17] utilize the nanoparticle core to arrange the linear nucleic acid components into functional forms. Indeed, it is the particle core that dictates the shape of the SNA, and to date, single-stranded and double-stranded versions of these materials have been created that consist of DNA, LNA, and RNA.

SNAs should also not be confused with their monovalent analogues – individual particles coupled to a single DNA strand.[18] Such single strand-nanoparticle conjugate structures have led to interesting advances in their own right, but do not exhibit the unique properties of SNAs.

Applications and Societal Benefit

Intracellular Gene Regulation
Figure 4. Nucleic acids arranged in a spherical geometry offer a fundamentally new path toward gene regulation. Benefits to this approach include the ability to enter cells without precomplexation with a transfection agent, nuclease resistance, and minimal immune response.[1]

SNAs have been proposed as nanotherapeutics.[13] Despite their high negative charge, these nanostructures are taken up by cells, without the need for positively charged polymeric co-carriers, and are effective as gene regulation agents in both siRNA and antisense gene regulation pathways (Fig. 4).[13][19] The proposed mechanism is that, unlike their linear counterparts, SNAs have the ability to complex scavenger proteins and thereby facilitate endocytosis. Investigation of the mechanism for SNA uptake[20] revealed that enzymatic degradation (nuclease) is reduced as compared to a linear oligonucleotide .[21] These materials have been suggested for a wide variety of therapeutic treatments which are developed by the company Exicure. The first clinical trial, in collaboration with Purdue Pharma, focused on SNA AST-005 for chronic plaque psoriasis. It showed no toxicity but also no significant efficacy and Purdue Pharma has notified Exicure it has declined to exercise its option to develop AST-005 at this time.[22]

SNAs packed gold nanoparticles are able to deliver small interfering RNA (siRNA) to treat glioblastoma multiforme in a proof-of-concept study using mouse model, reported by the research team led by Mirkin. The iRNA targets Bcl2Like12 gene overexpressed in glioblastoma tumors and silences the oncogene. The gold nanoparticles injected intravenously cross the blood-brain barrier and find their target in the brain. In the animal model, the treatment caused a 20% increase in survival rate and 3 to 4 fold reduction in tumor size. Mirkin indicated that this novel therapeutic approach establishes a platform for treating a wide range of diseases.[23][24]

Molecular Diagnostics
Figure 5. The FDA-cleared Verigene System, developed and commercialized by Nanosphere, Inc., a company spun-out of research projects initiated in Mirkin’s laboratory at Northwestern University. There are nine FDA-cleared panel assays based upon SNAs, now used by hospitals all over the world.

The Mirkin group has proposed spherical nucleic acids as novel labels for in vitro biodetection schemes and intracellular assays, and as potent cell transfection, therapeutic, and gene regulation materials.[13][25][26][27][28][29][30][31] The FDA-cleared Verigene System, commercialized by Nanosphere, is now sold in over twenty countries (Fig. 5). This technology allows the detection of markers for many diseases, including infectious disease and cancers, with a sensitivity and selectivity far exceeding that of conventional diagnostic tools. Indeed, the Verigene is transforming patient care by transitioning molecular diagnostic screening from centralized, often remote, analytical laboratories to the local hospital setting, which decreases the time required for diagnosis. Further, the Verigene has enabled the identification of new markers for Alzheimer's disease,[26] HIV,[32][33] and cardiac disease[34][35][36] as well as new tests for the early detection of a variety of forms of prostate cancer.[37] These medical diagnostic and therapeutic tools have already saved or improved many lives. As such, SNAs represent an important class of diagnostic probes that will enable fundamental discoveries and empower physicians to make quick and accurate decisions about patient care regarding almost any disease with a genetic basis.

Intracellular Probes
Figure 6. General schematic of NanoFlare-based detection.

NanoFlares utilize the SNA architecture for intracellular mRNA detection. In this design, alkanethiol-terminated antisense DNA strands (complementary to a target mRNA strand within cells) are attached to the surface of a gold nanoparticle. Fluorophore-labeled “reporter strands” are then hybridized to the SNA construct to form the NanoFlare. When the fluorophore labels are brought within close proximity of the gold surface, as controlled by programmable nucleic acid hybridization, their fluorescence is quenched (Fig. 6).[30] Following cellular entry (enabled by the SNA geometry), reporter strands are concomitantly driven from the NanoFlare and replaced by the longer, target mRNA sequence. Note that mRNA binding is thermodynamically favored since the strands holding the reporter sequence have greater overlap of their nucleotide sequence with the target mRNA. Upon reporter strand release, dye fluorescence is no longer quenched by the gold nanoparticle core, and increased fluorescence is observed. This method for RNA detection provides the only way to sort live cells based upon genetic content. AuraSense and AuraSense Therapeutics are two start-up companies founded to advance these SNA structures in several sectors of the biomedical industry. In 2011, AuraSense entered into partnership with EMD-Millipore to commercialize NanoFlares under the SmartFlare trade name. In 2015, there were over 1600 commercial forms of SmartFlares sold worldwide.[38] However, two years later, very few applications have been published and only the product is discontinued.[39] Furthermore, several scientific publications dispute the fact that the NanoFlares (SmartFlares) can detect mRNA and point out that the endosomal intracellular localization of Smartflares after uptake is not compatible with mRNA detection, and that the detection range does not match with intracellular concentrations of mRNA.[3][3][40][41]

Materials Synthesis

SNAs also have been utilized to develop an entirely new field of materials science – one that focuses on using SNAs as synthetically-programmable building blocks in the construction of DNA-nanoparticle superlattices (Fig. 7). In 2011, a landmark paper was published in Science that defines a set of design rules for making superlattice structures of tailorable crystallographic symmetry and lattice parameters with sub-nm precision.[42] The rules are described as follows:

Figure 7. Examples of the types of crystal structures that can be formed using design rules for preparing colloidal crystals. Note unit cell schematic, small angle x-ray scattering (SAXS) and electron microscopy data are shown for each example.

1. When all DNA-NPs in a system possess equal hydrodynamic radii, each NP in the thermodynamic product will maximize the number of nearest neighbors to which it can form DNA connections.

2. When two lattices are of similar stability, the kinetic product can be produced by slowing the rate at which individual DNA linkers dehybridize and subsequently rehybridize.

3. The overall hydrodynamic radius of a DNA-NP, rather than the sizes of its individual NP or oligonucleotide components, dicates its assembly and packing behavior.

4. In a binary system, the size ratio and DNA linker ratio between two particles dictate the thermodynamically favored crystal structure.

5. Two systems with the same size ratio and DNA linker ratio exhibit the same thermodynamic product.

6. The most stable crystal structure will maximize all possible types of DNA sequence-specific hybridization interactions.

These design rules are analogous to Pauling's rules for ionic crystals, but ultimately more powerful. For example, when using atomic or ionic building blocks in the construction of materials, the crystal structure, symmetry, and spacing are fixed by atomic radii and electronegativity. However, in the nanoparticle-based system, crystal structure can be tuned independent of the nanoparticle size and composition by simply adjusting the length and sequence of the attached DNA. This has enabled the construction of novel crystal structures for several materials systems and even crystal structures with no mineral equivalents.[43] The ability to place nanoparticles of any composition and shape at any location in a well-defined crystalline lattice with nm-scale precision should have far-reaching implications in areas ranging from catalysis to photonics to energy.

Economic Impact

The economic impact of SNA technology is substantial and rapidly growing. Three companies have been founded that are based on SNA technology - Nanosphere, AuraSense, and AuraSense Therapeutics (founded in 2000, 2009, and 2011, respectively). These companies have employed hundreds of people and commercialized over 1800 products. Nanosphere was one of the first nanotechnology-based biotechnology firms to go public in late 2007. The FDA-cleared Verigene System has been commercialized by Nanosphere, and nine accompanying FDA-cleared panel assays (Warfarin Metabolism, F5/F2/MTHFR, Influenza A and B, Staphylococcus aureus, 2009 H1N1) are now used by hospitals all over the world. Further, hundreds of research laboratories are currently utilizing these structures in all sorts of applications spanning genomics and proteomics.

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