Spherical Nucleic Acids

What are SNAs?

Spherical nucleic acids, or SNAs, (Figure 1) are three-dimensional nanostructures, typically consisting of densely functionalized and highly oriented nucleic acids covalently attached to the surfaces of spherical nanoparticle cores. Notably, these constructs define a new class of nucleic acids that have properties that markedly differ their linear cousin, on a sequence-for-sequence basis are able to enter cells efficiently to induce gene regulation (see video) and detect biological targets in live cells, as well as enable a number of other important biological applications. They also constitute fundamental building blocks in material science called programmable atom equivalents. Such structure, through established design rules, can be programmed to assembly into a wide variety of crystalline architectures.

Figure 1

(A) Existing structural forms of nucleic acids include linear duplexes, circular plasmid DNA, and three-dimensional SNA.
(B) Natural and synthetic assemblies of nucleic acids.

Spherical Nucleic Acids

This animation describes the structure, properties, and uses of the spherical nucleic acid (SNA), a construct typically composed of a nanoparticle core and densely packed and highly oriented nucleic acid shell. This structure has unique architectural-dependent properties that set it apart from all other forms of nucleic acids. These properties make SNAs extremely useful in in vitro medical diagnostics, intracellular detection, and therapeutics.

The Structure and Properties of SNA

The core of the spherical nucleic acid serves two purposes – it imparts the conjugate with novel chemical and physical properties, and it acts as a scaffold for assembling and orienting the oligonucleotides into a dense spherical arrangement that gives rise to many of their functional properties, distinguishing them from all other forms of matter.  Recent studies have shown that one can use the gold core as a scaffold, subsequently crosslink the DNA at the base of the particle, and dissolve the gold to create a crosslinked polymer-supported SNA without the inorganic core, which exhibits many of the hallmark properties of the original gold nanoparticle conjugate. This includes the ability to cooperatively hybridize with complementary nucleic acids and form stronger duplexes than the same sequence of linear DNA and the ability to efficiently transfect cell membranes without the need for co-carriers.

Up to now, we have developed a series of SNA structures with different types of cores, ranging from inorganic nanoparticles, to polymers and micelles and to biological components such as proteins (Figure 2). These studies underscored one of the fundamental features of SNAs – many of the properties of these nanomaterials stem from a dense layer of oriented nucleic acids and are core-independent. While most forms of nucleic acids rely on the hybridized duplex as the fundamental structural unit that determines their overall shape, SNAs can be prepared from both single- and double-stranded nucleic acids, and their orientation is determined by the shape of the inorganic core. SNA nanostructures are distinct from structures made by “DNA origami” – they can be synthesized independent of nucleic acid sequence and hybridization interactions and are formed via chemical bonds, not recognition processes.

Figure 2

Scheme showing many different types of SNA structures based on different cores.

Live Cell mRNA Detection: The Nanoflare

NanoFlares are a spherical nucleic acid constructs capable of detecting intracellular targets in live cells at the single-cell level (Figure 3). A NanoFlare typically consists of a spherical gold nanoparticle densely functionalized with a monolayer of single-stranded DNA (ssDNA) for target. The ssDNA “recognition sequence” is pre-hybridized to a shorter DNA complement containing a fluorescent reporter (the “reporter flare”) whose fluorescence is quenched based on its proximity to the gold particle. When target binds to the recognition sequence, the flare strand is displaced, providing a fluorescent readout.

These constructs have been shown to enter many different cell lines and primary cells tested to date, and they are believed to do so by engaging scavenger receptors that facilitate caveolin-mediated endocytosis. They resist nuclease degradation, lack cytotoxicity, and are effective as either single-gene or multigene detection agents, depending upon how they are formulated. The Nanoflare constructs have been used to detect metastatic markers such as Vimentin, Twist, and they can be used with whole blood samples to detect circulating tumor cells and diagnose patient risk for metastasis. They constitute the most effective way of measuring genetic content in live cells at the single cell level.

Figure 3

(A) Scheme showing the design and detection process of the NanoFlare.

(B) Fluoresce microscope images showing the detection of survivin mRNA in living cells using NanoFlares.

Ultrasensitive Biomarker Detection: Scanometric Assay

The scanometric assay are emerging diagnostic tools used for the enzyme-free ultrasensitive detection of various protein and nucleic acid targets. The detection methods are based upon the use of AuNP conjugates. The assay utilizes array slides to capture protein and nucleic acid targets and then sandwiches it with the AuNP probes. The signal is then amplified by catalytic reduction of Ag(I) or Au(III) in the presence of reducing agents. After the reduction step, the slide is used as a wave guide, and scattered light is measured from the metal spots to determine target identity and concentration (Figure 4). The utility of the scanometric assay are able to detect biomolecules, such as proteins and nucleic acids at extremely low concentrations. In the case of proteins such as PSA, the assay can reach the detection limit between one and six orders of magnitude more sensitive than conventional ELISA-based assays. In the case of nucleic acid detection, the LOD is 100 aM for large DNA targets and does not require PCR or related target amplification techniques. The scanometic assays have been also been used to profile the expression of miRNA species from human serum, cell culture, and human tissue samples.

Figure 4

Left: Scheme showing the miRNA profiling process using Scano-miR technology.

Right: Light-scattering images of miRNA profiling pattern on microarray.

SNA-based Therapeutic Technology

SNAs for Gene Regulation

Regulation of gene expression with synthetic oligonucleotides has led to fundamental breakthroughs in the understanding of intracellular function and may lead to viable treatment options for genetic-based diseases, such as many forms of cancer and neurological disorders. However, the delivery of synthetic nucleic acids to disease sites and across cell membranes is still a major challenge for gene regulation therapies (antisense DNA and siRNA). Indeed, Nature has created a defense network for foreign nucleic acids. For example, since nucleic acids are negatively charged, they cannot easily cross the negatively charged cell membrane.

Furthermore, they are rapidly degraded by nucleases and activate the innate immune response in cells. Historically, researchers have required the use of transfection agents to shuttle the nucleic acids through the negatively charged cellular membrane and shield them from enzymatic degradation. Unfortunately, these materials are not ideal for systemic delivery because of their inability to be degraded naturally, severe immunogenicity, and toxicity at high concentrations.

SNA constructs provide an alternative in this regard since, despite their large negative charge (zeta potential < −30 mV), they have been found to enter cells in very high numbers, without the need for ancillary transfection agents. Additionally, SNAs have a unique set of properties specific for intracellular applications, such as high binding coefficients for complementary DNA and RNA, nuclease resistance, minimal immune response, no observed toxicity, and highly effective gene regulating capabilities. Research within the group focuses on the mechinism of SNA trafficking within cells and tissues, as well as therapeutic applications that exploit these important observations.

SNAs for Immunodulation

Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized.

Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies. Projects within the group focus on developing a fundamental understanding of the pathways that can be engaged by IS-SNAs and new therapies for triple-negative breast cancer, melanoma and bladder cancer.

Figure 5

Cartoon representation of β-gal before (left) and after (right) functionalization with DNA. The representation was adapted from PDB ID 1BGL.32

Protein Transfection using SNAs

Proteins represent a highly evolved class of natural nanoparticles with an unparalleled degree of structural and compositional homogeneity, as well as diversity of functional applications. In particular, the efficient intracellular delivery of functional proteins has widespread potential in medicine and provides a means for engineering cellular functions. However, the cellular uptake of functional proteins is impeded by their inherent instability, large sizes, and charged surfaces. Recently, spherical nucleic acid (SNA)-nanoparticle conjugates, which consist of a nanoparticle core surrounded by a dense shell of oligonucleotides, have emerged as exciting new architectures with diverse biological applications in gene regulation, immunomodulation, and intracellular detection.

These applications are possible due to the superior cellular uptake and physiological stability of SNAs relative to their individual components. This enhanced cellular internalization of SNAs is derived from the 3-D architecture of the conjugates and its ability to engage scavenger receptors on the surfaces of most cells. Importantly, the favorable biological properties of SNAs are independent of their nanoparticle cores, which can therefore be chosen based on potential biological applications rather than practical synthetic limitations.

Based on these observations, we hypothesized that proteins could serve as the nanoparticle core of SNAs and the dense shell of oligonucleotides as a biocompatible polymer shell that promotes cellular uptake. These supramolecular structures, termed ProSNAs (Figure 5). ProSNAs are emerging as a new class of protein transfection materials composed of a functional protein core chemically modified with a dense shell of oligonucleotides. β-galactosidase ProSNAs retain the native structure and catalytic ability of the hydrolytic enzyme β-galactosidase, which serves as the protein core, despite the functionalization of its surface with ∼25 DNA strands. The covalent attachment of a shell of oligonucleotides to the surface of β-galactosidase enhances its cellular uptake by up to ∼280-fold and allows for the use of working concentrations as low as 100 pM enzyme. DNA-functionalized β-galactosidase retains its ability to catalyze the hydrolysis of β-glycosidic linkages once endocytosed, whereas equal concentrations of protein show little to no intracellular catalytic activity (Figure 6).

Figure 6

Intracellular catalytic activity of native and ProSNA β-gal. (A) Light micrographs of HaCaT (left), SKOV3 (middle), and C166 (right) cells after incubation with the β-gal substrate, Xgal. The blue color apparent in cells pretreated with ProSNA β-gal results from the hydrolysis of Xgal and formation of an insoluble reaction product. Scale bar = 100 μm.

Bio Subgroup Members

Members of the NanoBiology subgroup come from diverse backgrounds spanning biology, chemistry, materials science and engineering, biomedical engineering, and chemical and biological engineering. This subgroup works to understand the unique biological, chemical, and physical properties of spherical nucleic acids (SNAs) while engineering novel biodetection and nanotherapeutic approaches based upon these nanostructures.

Standing (L-R): Gokay Yamankurt (co-leader), Andrew Sinegra, Carlo Berti, Brian Meckes, Jungsoo Park, Robert Stawicki, Shengshuang Zhu, Isaac Larkin, Ziyin Huang, AJ Sprangers, Kacper Skakuj, Adam Ponedal, Hang Xing, Sasha Ebrahimi, Brandon Liauw, Nathan Kim

Sitting (L-R): Caroline Kusmierz, Wuliang Zhang, Lisa Cole (co-leader), Shuya Wang, Jen Ferrer, Precious Cantu