Jessica L. Rouge

Nucleotide-stabilized cadmium sulfide nanoparticles

Cadmium sulfide nanoparticles stabilized by both natural and unnatural 5′-nucleotide triphosphates were investigated to elucidate the specific chemical functionalities involved in the synthesis of emissive materials. The roles of the nucleobase functionalities in semiconductor nanocrystal synthesis are deconvoluted using photoluminescence spectroscopy, transmission electron microscopy and agarose gel electrophoresis. Through a survey of all the natural nucleosides, it was discovered that 5′-guanosine triphosphate most effectively stabilizes emissive nanoparticles, while adenosine, inosine, cytidine, uracil and 7-methylguanosine nucleobases do not facilitate successful synthesis of emissive product unless used under basic conditions. The work presented systematically explores and identifies important functionalities within polynucleic acids that can be used to produce aqueous soluble semiconductor nanocrystals.

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.

Biomolecules in the synthesis and assembly of materials for energy applications

Biomolecules such as RNA, DNA, peptides, and proteins are emerging as powerful chemical tools for the synthesis of inorganic nanoparticles. Specific biomolecule sequences have been isolated that afford remarkable control over the size, shape, polymorph, and hierarchical assembly of nanoparticles. Such exquisite control over nanoparticle growth and integration has already produced materials with unexpected photophysical properties and battery devices with improved performance. Continued exploration of biomolecule-mediated materials synthesis portends further advances in materials for the energy sciences. This review surveys the use of biomolecules in the synthesis and assembly of materials with a primary focus on methods that allow vast landscapes of biomolecule sequence space to be sampled simultaneously to discover unique sequence codes for new materials.

Nucleic Acid Nanocapsules for Enzyme-Triggered Drug Release

Herein we describe a nucleic acid functionalized nanocapsule in which nucleic acid ligands are assembled and disassembled in the presence of enzymes. The particles are fully degradable in response to esterases due to an embedded ester cross-linker in the particles core. During synthesis the nanocapsules can be loaded with hydrophobic small molecules and post self-assembly undergo covalent cross-linking using copper catalyzed click chemistry. They can then be functionalized with thiolated DNA through stepwise thiolyne chemistry using UV light irradiation. Additionally, the capsule is compatible with enzyme mediated functionalization of a therapeutic mRNA-cleaving DNAzyme at the particles surface. The resulting particle is highly stable, monodisperse in size, and maximizes the therapeutic potential of both the particles interior and exterior.

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.

Tunable DNA cleavage by intercalating peptidoconjugates.

The properties of a novel family of peptide‐based DNA‐cleavage agents are described. Examination of the DNA‐cleavage activities of a systematic series of peptide–intercalator conjugates revealed trends that show a strong dependence on peptide sequence. Conjugates differing by a single residue displayed reactivities that varied over a wide range. The cleavage activity was modulated by the electrostatic or steric qualities of individual amino acids. Isomeric conjugates that differed in the position of the tether also exhibited different reactivities. The mechanism of DNA cleavage for these compounds was also probed and was determined to involve hydrogen‐atom abstraction from the DNA backbone. Previous studies of these compounds indicated that amino acid peroxides were the active agents in the cleavage reaction; in this report, the chemistry underlying the reaction is characterized. The results reported provide insight into how peptide sequences can be manipulated to produce biomimetic compounds.

Single molecule studies of quantum dot fluorescence intermittency: evidence for both dark and light-assisted blinking dynamics

The present work investigates single CdSe QD blinking behaviour in the absence of illumination, in order to elucidate the role of photoexcitation on the long time dynamics. The probability of a QD transitioning in the dark from a high quantum yield (‘on’) state to a low quantum yield (‘off’) state is found to be extremely low and essentially independent of dwell time from <100 ms to minute time scales. The absence of a dark pathway is consistent with many models, for example, in which ‘on’ to ‘off’ blinking is ascribed to photo-assisted Auger ionization and charging of the QD. On the other hand, blinking recovery statistics (from ‘off’ to ‘on’ state) reveal an approximate power law dependence on dwell time, consistent with non-photoactivated and highly distributed kinetics in the dark. Most importantly, such dark QD recovery is orders of magnitude slower than predicted by previous power law kinetic studies under constant illumination conditions, highlighting the presence of both (i) light-activated and (ii) dark blinking recovery pathways. Furthermore, the kinetic analysis indicates that nearly all single CdSe QD studies to date have been performed under conditions where blinking recovery is dominated by the light-activated channel to date.

Cooperativity between two selected RNA Pdases in the synthesis of Pd nanoparticles

We have made the surprising discovery that the crystallinity of nanoparticles formed from solutions containing RNA depends upon the presence of sequence mixtures. That is, a single sequence selected from the original random RNA sequence library produced mostly amorphous hexagonal nanoparticles, while a combination of sequences that emerged from the selection yields crystalline material as determined by SAED. To our knowledge this is the first example in which two biomolecules (RNA, DNA, or peptides) selected in vitro work together to provide a unique chemical outcome. In addition, this article provides a rigorous examination of the chemistry of Pd nanoparticle formation using RNA and the organometallic precursor complex Pd2(DBA)3 (DBA is dibenzylideneacetone). These studies have identified the specific conditions required for the successful RNA-mediated synthesis of Pd nanoparticles from aqueous solutions (10% THF : 90% H2O) containing Pd2(DBA)3, as well as conditions that led to anomalous results. A variety of techniques were employed to characterize materials formed under different solution conditions including SEM, AFM, TEM, selected area electron diffraction (SAED), and a chemical reactivity test. These analysis methods support the formation of Pd particles by RNA mediation when accounting for and controlling the important variables in the execution of the experiments. It is now clear that nanoparticles formed from RNA sequences isolated viain vitro selection can be dependent on many factors and it is understood that the specific sequence or sequence mixtures must be taken into account to fully understand RNA mediation of nanoparticle formation.

Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a Modular Platform for Enzyme Specific Cargo Release

Herein we describe a modular assembly strategy for photo-cross-linking peptides into nucleic acid functionalized nanocapsules. The peptides embedded within the nanocapsules form discrete nanoscale populations capable of gating the release of molecular and nanoscale cargo using enzyme-substrate recognition as a triggered release mechanism. Using photocatalyzed thiol-yne chemistry, different peptide cross-linkers were effectively incorporated into the nanocapsules and screened against different proteases to test for degradation specificity both in vitro and in cell culture. By using a combination of fluorescence assays, confocal and TEM microscopy, the particles were shown to be highly specific for their enzyme targets, even between enzymes of similar protease classes. The rapid and modular nature of the assembly strategy has the potential to be applied to both intracellular and extracellular biosensing and drug delivery applications.

Enzymatically Ligated DNA-Surfactants: Unmasking Hydrophobically Modified DNA for Intracellular Gene Regulation

Herein, we describe the characterization of a novel self‐assembling and intracellular disassembling nanomaterial for nucleic acid delivery and targeted gene knockdown. By using a recently developed nucleic acid nanocapsule (NAN) formed from surfactants and conjugated DNAzyme (DNz) ligands, it is shown that DNz–NAN can enable cellular uptake of the DNAzyme and result in 60 % knockdown of a target gene without the use of transfection agents. The DNAzyme also exhibits activity without chemical modification, which we attribute to the underlying nanocapsule design and release of hydrophobically modified nucleic acids as a result of enzymatically triggered disassembly of the NAN. Fluorescence‐based experiments indicate that the surfactant‐conjugated DNAzymes are better able to access a fluorescent mRNA target within a mock lipid bilayer system than the free DNAzyme, highlighting the advantage of the hydrophobic surfactant modification to the nucleic acid ligands. In vitro characterization of DNz–NANs substrate‐cleavage kinetics, stability in biological serum, and persistence of knockdown against a proinflammatory transcription factor, GATA‐3, are presented.