Nanoparticles of a metal-organic framework (MOF), UiO-66-N3 (Zr6O4OH4(C8H3O4-N3)6), were synthesized. The surface of the MOF was covalently functionalized with oligonucleotides, utilizing a strain promoted click reaction between DNA appended with dibenzylcyclooctyne and azide-functionalized UiO-66-N3 to create the first MOF nanoparticle-nucleic acid conjugates. The structure of the framework was preserved throughout the chemical transformation, and the surface coverage of DNA was quantified. Due to the small pore sizes, the particles are only modified on their surfaces. When dispersed in aqueous NaCl, they exhibit increased stability and enhanced cellular uptake when compared with unfunctionalized MOF particles of comparable size.

We report the development of the multiplexed nano-flare, a nanoparticle agent that is capable of simultaneously detecting two distinct messenger RNA (mRNA) targets inside a living cell. These probes consist of polyvalent DNA-functionalized gold nanoparticles with multiple DNA sequences, each hybridized to a reporter with a distinct fluorophore label, and each complementary to its corresponding mRNA target. When multiplexed nano-flares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nano-flares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.

We report a novel spherical nucleic acid (SNA) gold nanoparticle conjugate, termed the Sticky-flare, which enables facile quantification of RNA expression in live cells and spatiotemporal analysis of RNA transport and localization. The Sticky-flare is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence-specific manner. On recognition, the Sticky-flare transfers a fluorophore-conjugated reporter to the transcript, resulting in a turning on of fluorescence in a quantifiable manner and the fluorescent labeling of targeted transcripts. The latter allows the RNA to be tracked via fluorescence microscopy as it is transported throughout the cell. We use this novel nanoconjugate to analyze the expression level and spatial distribution of β-actin mRNA in HeLa cells and to observe the real-time transport of β-actin mRNA in mouse embryonic fibroblasts. Furthermore, we investigate the application of Stickyflares for tracking transcripts that undergo more extensive compartmentalization by fluorophore-labeling U1 small nuclear RNA and observing its distribution in the nucleus of live cells.he study of RNA is a critical component of biological research and in the diagnosis and treatment of disease. Recently, the localization of mRNA has been identified as an essential process for a number of cellular functions, including restricting the production of certain proteins to specific compartments within cells (1). For instance, synaptic potentiation, the basis of learning and memory, relies on the local translation of specific mRNAs in pre-and postsynaptic compartments (2). Likewise, the misregulation of RNA distribution is associated with many disorders, including mental retardation, autism, and cancer metastasis (3-5). However, despite the significant role of mRNA transport and localization in cellular function, the available methods to visualize these phenomena are severely limited. For example, FISH, the most commonly used technique to analyze spatial distribution of RNA, requires fixation and permeabilization of cells before analysis (6). As a result, analysis of dynamic RNA distribution is restricted to a single snapshot in time (7,8). With such a limitation, understanding the translocation of RNA with respect to time, cell cycle, or external stimulus is difficult if not impossible. Furthermore, fixed cell analysis is a lengthy and highly specialized procedure due to the number of steps necessary to prepare a sample. Fixation, permeabilization, blocking, and staining processes each require optimization and vary based on cell type and treatment conditions, rendering FISH prohibitively complicated in many cases. Likewise, live cell analysis platforms such as molecular beacons require toxic transfection techniques, such as microinjection or lipid transfection, and are rapidly sequestered to the nucleus on cellular entry (9, 10). Recently more sophisticated live cell analyses have been developed that use genetic engineering to introduce exogenous hybrid gene...

Ribozymes are highly structured RNA sequences that can be tailored to recognize and cleave specific stretches of mRNA. Their current therapeutic efficacy remains low due to their large size and structural instability compared to shorter therapeutically relevant RNA such as small interfering RNA (siRNA) and microRNA (miRNA). Herein, a synthetic strategy that makes use of the spherical nucleic acid (SNA) architecture to stabilize ribozymes and transfect them into live cells is reported. The properties of this novel ribozyme SNA are characterized in the context of the targeted knockdown of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein involved in chemotherapeutic resistance of solid tumors, foremost glioblastoma multiforme (GBM). Data showing the direct cleavage of full-length MGMT mRNA, knockdown of MGMT protein, and increased sensitization of GBM cells to therapy-mediated apoptosis, independent of transfection agents, provide compelling evidence for the promising properties of this new chemical architecture.

Herein, we describe a rapid, divergent method for using spherical nucleic acids (SNAs) as a universal platform for attaching RNA to DNA-modified nanoparticles using enzyme-mediated techniques. This approach provides a sequence-specific method for the covalent attachment of one or more in vitro transcribed RNAs to a universal SNA scaffold, regardless of RNA sequence. The RNA–nanoparticle constructs are shown to effectively knock down two different gene targets using a single, dual-ligated nanoparticle construct.