dna-tethered-rna-polymerase-for-programmable-vitro-transcription

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

University of Toronto

Quantum dot (QD) based contrast agents are currently being developed as probes for bioimaging and as vehicles for drug delivery. The ability to detect QDs, regardless of fluorescence brightness, in cells, tissues, and organs is imperative to their development. Traditional methods used to visualize the distribution of QDs in biological samples mainly rely on fluorescence imaging, which does not account for optically degenerate QDs as a result of oxidative quenching within the biological environment. Here, we demonstrate the use of silver staining for directly visualizing the distribution of QDs within biological samples under bright field microscopy. This strategy involves silver deposition onto the surface of QDs upon reduction by hydroquinone, effectively amplifying the size of QDs until visible for detection. The method can be used to detect non-fluorescent QDs and is fast, simple, and inexpensive.

Visualizing quantum dots in biological samples using silver staining.

The ability to target contrast agents and therapeutics inside cells is becoming important as we strive to decipher the complex network of events that occur within living cells and design therapies that can modulate these processes. Nanotechnology researchers have generated a growing list of nanoparticles designed for such applications. These particles can be assembled from a variety of materials into desirable geometries and configurations and possess useful properties and functionalities. Undoubtedly, the effective delivery of these nanomaterials into cells will be critical to their applications. In this tutorial review, we discuss the fundamental challenges of delivering nanoparticles into cells and to the targeted organelles, and summarize strategies that have been developed to-date.

Strategies for the intracellular delivery of nanoparticles.

We aim to develop a facile strategy for assembling nanoparticles within cross-linked polymer micelles that enables tuning of their overall hydrodynamic size and surface charge and to mitigate toxicity.

A strategy to assemble nanoparticles with polymers for mitigating cytotoxicity and enabling size tuning.

Quantum dots that contain cadmium, selenium and zinc are not toxic to monkeys for periods of up to 90 days, but longer-term studies are needed to determine the ultimate fate of the heavy metals that accumulate in the organs.

Nanotoxicology. No signs of illness.

The assembly of nanomaterials using DNA can produce complex nanostructures, but the biological applications of these structures remain unexplored. Here, we describe the use of DNA to control the biological delivery and elimination of inorganic nanoparticles by organizing them into colloidal superstructures. The individual nanoparticles serve as building blocks, whose size, surface chemistry and assembly architecture dictate the overall superstructure design. These superstructures interact with cells and tissues as a function of their design, but subsequently degrade into building blocks that can escape biological sequestration. We demonstrate that this strategy reduces nanoparticle retention by macrophages and improves their in vivo tumour accumulation and whole-body elimination. Superstructures can be further functionalized to carry and protect imaging or therapeutic agents against enzymatic degradation. These results suggest a different strategy to engineer nanostructure interactions with biological systems and highlight new directions in the design of biodegradable and multifunctional nanomedicine. 

DNA assembly of nanoparticle superstructures for controlled biological delivery and elimination.

DNA assembly of nanoparticles is a powerful approach to control their properties and prototype new materials. However, the structure and properties of DNA-assembled nanoparticles are labile and sensitive to interactions with counterions, which vary with processing and application environment. Here we show that substituting polyamines in place of elemental counterions significantly enhanced the structural rigidity and plasmonic properties of DNA-assembled metal nanoparticles. These effects arose from the ability of polyamines to condense DNA and cross-link DNA-coated nanoparticles. We further used polyamine wrapped DNA nanostructures as structural templates to seed the growth of polymer multilayers via layer-by-layer assembly, and controlled the degree of DNA condensation, plasmon coupling efficiency, and material responsiveness to environmental stimuli by varying polyelectrolyte composition. These results highlight counterion engineering as a versatile strategy to tailor the properties of DNA-nanoparticle assemblies for various applications, and should be applicable to other classes of DNA nanostructures. 

Engineering the Structure and Properties of DNA-Nanoparticle Superstructures Using Polyvalent Counterions.

The use of DNA to assemble inorganic nanoparticles into superstructures is an emerging strategy to build non-toxic delivery vehicles for targeting diseases in the body. The impact of the core-satellite nanosystem design in mediating drug storage, drug release (via heat), and killing of HeLa cells in culture is investigated.

Tuning the Drug Loading and Release of DNA-Assembled Gold-Nanorod Superstructures.

Understanding the interaction of molecularly assembled nanoparticles with physiological fluids is critical to their use for in vivo delivery of drugs and contrast agents. Here, we systematically investigated the factors and mechanisms that govern the degradation of DNA on the nanoparticle surface in serum. We discovered that a higher DNA density, shorter oligonucleotides, and thicker PEG layer increased protection of DNA against serum degradation. Oligonucleotides on the surface of nanoparticles were highly resistant to DNase I endonucleases, and degradation was carried out exclusively by protein-mediated exonuclease cleavage and full-strand desorption. These results enabled the programming of the degradation rates of the DNA-assembled nanoparticle system from 0.1 to 0.7 h and the engineering of superstructures that can release two different preloaded dye molecules with distinct kinetics and half-lives ranging from 3.3 to 9.8 h. This study provides a general framework for investigating the serum stability of DNA-containing nanostructures. The results advance our understanding of engineering principles for designing nanoparticle assemblies with controlled in vivo behavior and present a strategy for storage and multistage release of drugs and contrast agents that can facilitate the diagnosis and treatment of cancer and other diseases.

Controlling DNA-nanoparticle serum interactions.

Advances in the field of structural DNA nanotechnology have produced a growing number of nanostructures that are now being developed for diverse applications. Often, these nanostructures contain not only nucleic acids but also a myriad of other classes of molecules and materials such as proteins, lipids, sugars, and synthetic polymers. Increasing structural and compositional complexity promises new functional capabilities, but also demands new tools for design verification. Systematically verifying the design of DNA-scaffolded nanomaterials is necessary to identify and to refine their design rules, and to enable the field to progress toward "real world" applications. In this issue of , Bertosin  used single-particle cryo-electron microscopy to characterize the structure of multilayer DNA origamis following coating with oligolysine-based polymers, a class of material which has previously been shown to stabilize DNA nanostructures in physiological environments for use in biological applications. This Perspective summarizes their findings, discusses the broader challenges of verifying the design of DNA nanotechnologies incorporating complex materials, and highlights future directions for advancing their applications.

Design Verification as Foundation for Advancing DNA Nanotechnology Applications.

Immune cells sense, communicate, and logically integrate a multitude of environmental signals to make important cell-fate decisions and fulfill their effector functions. These processes are initiated and regulated by a diverse array of immune receptors and via their dynamic spatiotemporal organization upon ligand binding. Given the widespread relevance of the immune system to health and disease, there have been significant efforts toward understanding the biophysical principles governing immune receptor signaling and activation, as well as the development of biomaterials which exploit these principles for therapeutic immune engineering. Here, how advances in the field of DNA nanotechnology constitute a growing toolbox for further pursuit of these endeavors is discussed. Key cellular players involved in the induction of immunity against pathogens or diseased cells are first summarized. How the ability to design DNA nanostructures with custom shapes, dynamics, and with site-specific incorporation of diverse guests can be leveraged to manipulate the signaling pathways that regulate these processes is then presented. It is followed by highlighting emerging applications of DNA nanotechnology at the crossroads of immune engineering, such as in vitro reconstitution platforms, vaccines, and adjuvant delivery systems. Finally, outstanding questions that remain for further advancing immune-modulatory DNA nanodevices are outlined.

Engineering DNA Nanostructures to Manipulate Immune Receptor Signaling and Immune Cell Fates.

The guiding principle for mineralized tissue formation is that mineral growth occurs through the interaction of Ca and phosphate ions with extracellular matrix (ECM) proteins. Recently, nanoengineered DNA structures have been proposed as mimics to ECM scaffolds. However, these principles have not been applied to mineralized tissues. Here, we describe DNA nanostructures, namely, a DNA nanotube and a DNA origami rectangle that are site specifically functionalized with a mineral-promoting "SSEE" peptide derived from ECM proteins present in mineralized tissues. In the presence of Ca and phosphate ions (mineralizing conditions), site-specific calcium phosphate formation occurred on the DNA nanostructures. Amorphous calcium phosphate or hydroxyapatite was formed depending on the incubation time, shape of the DNA nanostructure, and amount of Ca and phosphate ions present. The ability to design and control the growth of hydroxyapatite through nanoengineered scaffolds provides insights into the mechanisms that may occur during crystal nucleation and growth of mineralized tissues and can inspire mineralized tissue regeneration strategies.

Leo Y. T. Chou

Institute of Biomedical Engineering

University of Toronto

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

Leo Y. T. Chou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Biomedical Engineering

University of Toronto

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

Institute of Biomedical Engineering