Quantification and real-time tracking of RNA in live cells using Sticky-flares.

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 ß-actin mRNA in HeLa cells and to observe the real-time transport of ß-actin mRNA in mouse embryonic fibroblasts. Furthermore, we investigate the application of Sticky-flares 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.

Ribozyme-Spherical Nucleic Acids.

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 O(6)-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.

Nanoflares as probes for cancer diagnostics.

Patients whose cancer is detected early are much more likely to have a positive prognosis and outcome. Nanoflares hold promise as a practical diagnostic platform for the early detection of cancer markers in living cells. These probes are based on spherical nucleic acid (SNAs) and are typically composed of gold nanoparticle cores and densely packed and highly oriented oligonucleotide shells; these sequences are complementary to specific mRNA targets and are hybridized to fluorophore-labeled reporter strands. Nanoflares take advantage of the highly efficient fluorescence quenching properties of gold, the rapid cellular uptake of SNAs that occurs without the use of transfection agents, and the enzymatic stability of such constructs to report a highly sensitive and specific signal in the presence of intracellular target mRNA. In this chapter, we will focus on the synthesis, characterization, and diagnostic applications of nanoflares as they relate to cancer markers.

Biocompatible infinite-coordination-polymer nanoparticle-nucleic-acid conjugates for antisense gene regulation.

Herein, we report the synthesis of DNA-functionalized infinite-coordination-polymer (ICP) nanoparticles as biocompatible gene-regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3-hydroxy-4-pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe(III) to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe(III)-HOPO ICP particles by copper-free click chemistry afforded colloidally stable nucleic-acid nanoconstructs. The DNA-ICP particles, when cross-linked through sequence-specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA-ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.

Nucleic acid-metal organic framework (MOF) nanoparticle conjugates.

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.

Gene delivery to overcome astrocyte inhibition of axonal growth: an in vitro model of the glial scar.

After injury to the central nervous system, a glial scar develops that physically and biochemically inhibits axon growth. In the scar, activated astrocytes secrete inhibitory extracellular matrix, of which chondroitin sulfate proteoglycans (CSPGs) are considered the major inhibitory component. An inhibitory interface of CSPGs forms around the lesion and prevents axons from traversing the injury, and decreasing CSPGs can enhance axon growth. In this report, we established an in vitro interface model of activated astrocytes and subsequently investigated gene delivery as a means to reduce CSPG levels and enhance axon growth. In the model, a continuous interface of CSPG producing astrocytes was created with neurons seeded opposite the astrocytes, and neurite crossing, stopping, and turning were evaluated as they approached the interface. We investigated the efficacy of lentiviral delivery to degrade or prevent the synthesis of CSPGs, thereby removing CSPG inhibition of neurite growth. Lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase-1, decreased CSPGs, and reduced inhibition by the interface. Degradation of CSPGs by lentiviral delivery of chondroitinase also resulted in less inhibition and more neurites crossing the interface. These results indicate that the interface model provides a tool to investigate interventions that reduce inhibition by CSPGs, and that gene delivery can be effective in promoting neurite growth across an interface of CSPG producing astrocytes.

Multiplexed nanoflares: mRNA detection in live cells.

We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares 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 nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.

Analysis of mutations in tumor and normal adjacent tissue via fluorescence detection.

Inflammation is a major risk factor for many types of cancer, including colorectal. There are two fundamentally different mechanisms by which inflammation can contribute to carcinogenesis. First, reactive oxygen and nitrogen species (RONS) can damage DNA to cause mutations that initiate cancer. Second, inflammatory cytokines and chemokines promote proliferation, migration, and invasion. Although it is known that inflammation-associated RONS can be mutagenic, the extent to which they induce mutations in intestinal stem cells has been little explored. Furthermore, it is now widely accepted that cancer is caused by successive rounds of clonal expansion with associated de novo mutations that further promote tumor development. As such, we aimed to understand the extent to which inflammation promotes clonal expansion in normal and tumor tissue. Using an engineered mouse model that is prone to cancer and within which mutant cells fluoresce, here we have explored the impact of inflammation on de novo mutagenesis and clonal expansion in normal and tumor tissue. While inflammation is strongly associated with susceptibility to cancer and a concomitant increase in the overall proportion of mutant cells in the tissue, we did not observe an increase in mutations in normal adjacent tissue. These results are consistent with opportunities for de novo mutations and clonal expansion during tumor growth, and they suggest protective mechanisms that suppress the risk of inflammation-induced accumulation of mutant cells in normal tissue.

Spherical nucleic acids as a divergent platform for synthesizing RNA-nanoparticle conjugates through enzymatic ligation.

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.