A practical guide to time-resolved fluorescence microscopy and spectroscopy.

Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics across many timescales. With TCSPC, Fluorescence correlation spectroscopy (FCS) and pulsed interleaved excitation-Förster resonance energy transfer (PIE-FRET) are collected simultaneously on diffusing molecules to extract diffusion characteristics and proximity information. This article is a guide to calibrating FCS and PIE-FRET measurements with several biological samples including liposomes, streptavidin-coated quantum dots, proteins, and nucleic acids for reliable determination of diffusion coefficients and FRET efficiency. The FRET efficiency results are also compared to surface-attached single molecules using fluorescence lifetime imaging microscopy (FLIM-FRET). Combining the methods is a powerful approach to revealing mechanistic details of biological processes and pathways.

Demonstrating the Synthesis and Antibacterial Properties of Nanostructured Silver.

Investigating and understanding novel antibacterial agents is a necessary task as there is a constant increase in the number of multidrug-resistant bacterial species. The use of nanotechnology to combat drug-resistant bacteria is an important research area. The laboratory experiment described herein demonstrates that changes in the nanostructure of a material lead to significantly different antibacterial efficacies. Silver has been known to be an effective antibacterial agent throughout history, but its therapeutic uses are limited when present as either the bulk material or cations in solution. Silver nanoparticles (AgNPs) and DNA-templated silver nanoclusters (DNA-AgNCs) are both nanostructured silver materials that show vastly different antibacterial activities when incubated with E. coli in liquid culture. This work aims to provide students with hands-on experience in the synthesis and characterization of nanomaterials and basic microbiology skills; moreover, it is applicable to undergraduate and graduate curricula.

Structural Characterization of Nucleic Acid Nanoparticles Using SAXS and SAXS-Driven MD.

Structural characterization of nucleic acid nanoparticles (NANPs) in solution is critical for validation of correct assembly and for quantifying the size, shape, and flexibility of the construct. Small-angle X-ray scattering (SAXS) is a well-established method to obtain structural information of particles in solution. Here, we present a procedure for the preparation of NANPs for SAXS. This procedure outlines the steps for a successful SAXS experiment and the use of SAXS-driven molecular dynamics to generate an ensemble of structures that best explain the data observed in solution. We use an RNA NANP as an example, so the reader can prepare the sample for data collection, analyze the results, and perform SAXS-driven MD on similar NANPs.

Aptamer Conjugated RNA/DNA Hybrid Nanostructures Designed for Efficient Regulation of Blood Coagulation.

Disruptions to the hemostatic pathway can cause a variety of serious or even life-threatening complications. Situations in which the coagulation of blood has become disturbed necessitate immediate care. Thrombin-binding aptamers are single-stranded nucleic acids that bind to thrombin with high specificity and affinity. While they can effectively inhibit thrombin, they suffer from rapid degradation and clearance in vivo. These issues are resolved, however, by attaching the therapeutic aptamer to a nucleic acid nanostructure. The increased size of the nanostructure-aptamer complex elongates the post-infusion half-life of the aptamer. These complexes are also immunoquiescent. A significant benefit of using nucleic acids as anticoagulants is their rapid deactivation by the introduction of a nanostructure made fully from the reverse complement of the therapeutically active nanostructure. These advantages make nanoparticle conjugated antithrombin aptamers a promising candidate for a rapidly reversible anticoagulant therapy.

Synthesis of DNA-Templated Silver Nanoclusters and the Characterization of Their Optical Properties and Biological Activity.

DNA-templated silver nanoclusters (DNA-AgNCs) are a unique class of bioinorganic nanomaterials. The optical properties and biological activities of DNA-AgNCs are readily modulated by the minor adjustments in the sequence or structure of the templating oligonucleotide. Excitation-emission matrix spectroscopy (EEMS) enables the fluorescence of compounds to be measured in a way that examines the entirety of a material's fluorescent properties. The use of EEMS for the characterization of DNA-AgNCs allows for multiple fluorescence peaks to be readily identified while providing the excitation and emission wavelengths of each signal. To assess the antibacterial and cytotoxic activities of DNA-AgNCs, two separate experimental approaches are used. Assessing the growth of bacteria over time is accomplished by measuring the optical density of the bacterial suspension with 600 nm light, which is directly related to the number of bacteria in suspension. In order to evaluate the DNA-AgNCs for cytotoxic activity, cell viability assays which probe mitochondrial activity were used. Herein, we describe protocols for the characterization of the fluorescent, antibacterial, and cytotoxic activities of DNA-AgNCs using EEM, optical density measurements, and cell viability assays.

Optical, structural, and biological properties of silver nanoclusters formed within the loop of a C-12 hairpin sequence.

Silver nanoclusters (AgNCs) are the next-generation nanomaterials representing supra-atomic structures where silver atoms are organized in a particular geometry. DNA can effectively template and stabilize these novel fluorescent AgNCs. Only a few atoms in size - the properties of nanoclusters can be tuned using only single nucleobase replacement of C-rich templating DNA sequences. A high degree of control over the structure of AgNC could greatly contribute to the ability to fine-tune the properties of silver nanoclusters. In this study, we explore the properties of AgNCs formed on a short DNA sequence with a C12 hairpin loop structure (AgNC@hpC12). We identify three types of cytosines based on their involvement in the stabilization of AgNCs. Computational and experimental results suggest an elongated cluster shape with 10 silver atoms. We found that the properties of the AgNCs depend on the overall structure and relative position of the silver atoms. The emission pattern of the AgNCs depends strongly on the charge distribution, while all silver atoms and some DNA bases are involved in optical transitions based on molecular orbital (MO) visualization. We also characterize the antibacterial properties of silver nanoclusters and propose a possible mechanism of action based on the interactions of AgNCs with molecular oxygen.

Optical, structural and antibacterial properties of silver nanoparticles and DNA-templated silver nanoclusters.

Silver nanoparticles (AgNPs) are increasingly considered for biomedical applications as drug-delivery carriers, imaging probes and antibacterial agents. Silver nanoclusters (AgNCs) represent another subclass of nanoscale silver. AgNCs are a promising tool for nanomedicine due to their small size, structural homogeneity, antibacterial activity and fluorescence, which arises from their molecule-like electron configurations. The template-assisted synthesis of AgNCs relies on organic molecules that act as polydentate ligands. In particular, single-stranded nucleic acids reproducibly scaffold AgNCs to provide fluorescent, biocompatible materials that are incorporable in other formulations. This mini review outlines the design and characterization of AgNPs and DNA-templated AgNCs, discusses factors that affect their physicochemical and biological properties, and highlights applications of these materials as antibacterial agents and biosensors.

Biomotors, viral assembly, and RNA nanobiotechnology: Current achievements and future directions.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure-function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields.

Locking and Unlocking Thrombin Function Using Immunoquiescent Nucleic Acid Nanoparticles with Regulated Retention In Vivo.

The unbalanced coagulation of blood is a life-threatening event that requires accurate and timely treatment. We introduce a user-friendly biomolecular platform based on modular RNA-DNA anticoagulant fibers programmed for reversible extracellular communication with thrombin and subsequent control of anticoagulation via a "kill-switch" mechanism that restores hemostasis. To demonstrate the potential of this reconfigurable technology, we designed and tested a set of anticoagulant fibers that carry different thrombin-binding aptamers. All fibers are immunoquiescent, as confirmed in freshly collected human peripheral blood mononuclear cells. To assess interindividual variability, the anticoagulation is confirmed in the blood of human donors from the U.S. and Brazil. The anticoagulant fibers reveal superior anticoagulant activity and prolonged renal clearance in vivo in comparison to free aptamers. Finally, we confirm the efficacy of the "kill-switch" mechanism in vivo in murine and porcine models.

Expanding Structural Space for Immunomodulatory Nucleic Acid Nanoparticles (Nanps) via Spatial Arrangement of Their Therapeutic Moieties.

Different therapeutic nucleic acids (TNAs) can be unified in a single structure by their elongation with short oligonucleotides designed to self-assemble into nucleic acid nanoparticles (NANPs). With this approach, therapeutic cocktails with precisely controlled composition and stoichiometry of active ingredients can be delivered to the same diseased cells for enhancing pharmaceutical action. In this work, an additional nanotechnology-based therapeutic option that enlists a biocompatible NANP-encoded platform for their controlled patient-specific immunorecognition is explored. For this, a set of representative functional NANPs is extensively characterized in vitro, ex vivo, and in vivo and then further analyzed for immunostimulation of human peripheral blood mononuclear cells freshly collected from healthy donor volunteers. The results of the study present the advancement of the current TNA approach toward personalized medicine and offer a new strategy to potentially address top public health challenges related to drug overdose and safety through the biodegradable nature of the functional platform with immunostimulatory regulation.

DNA-Templated Fluorescent Silver Nanoclusters Inhibit Bacterial Growth While Being Non-Toxic to Mammalian Cells.

Silver has a long history of antibacterial effectiveness. The combination of atomically precise metal nanoclusters with the field of nucleic acid nanotechnology has given rise to DNA-templated silver nanoclusters (DNA-AgNCs) which can be engineered with reproducible and unique fluorescent properties and antibacterial activity. Furthermore, cytosine-rich single-stranded DNA oligonucleotides designed to fold into hairpin structures improve the stability of AgNCs and additionally modulate their antibacterial properties and the quality of observed fluorescent signals. In this work, we characterize the sequence-specific fluorescence and composition of four representative DNA-AgNCs, compare their corresponding antibacterial effectiveness at different pH, and assess cytotoxicity to several mammalian cell lines.

Tuning properties of silver nanoclusters with RNA nanoring assemblies.

Combining atomically resolved DNA-templated silver nanoclusters (AgNCs) with nucleic acid nanotechnology opens new exciting possibilities for engineering bioinorganic nanomaterials with uniquely tunable properties. In this unforeseen cooperation, nucleic acids not only drive the formation of AgNCs but also promote their spatial organization in supra-assemblies. In this work, we confirm the feasibility of this approach using programmable RNA rings to control formation and optical properteis of six individual AgNCs. "Red" (λEXC/λEM = 565/623 nm) and "green" (λEXC/λEM = 440/523 nm) emitting AgNCs are templated on cytosine-rich DNA fragments embedded into the RNA rings. Optical properties of the AgNCs formed on the RNA rings are characterized in detail. While all "red" species passively transition to "green" emitters with time, the initial fluorescent properties and relative stabilities of "red" AgNCs can be regulated by altering the relative orientation of AgNCs within the RNA rings. As such, the oxidative stability increases dramatically for AgNC positioned towards the center of the RNA rings rather than facing outward. Overall, our findings expand the existing AgNC fluorescent toolkit while uncovering the complexity of the AgNC electronic structures with the abundance of possibilities for controlling de-excitation processes.

Challenges to optimizing RNA nanostructures for large scale production and controlled therapeutic properties.

Nucleic acids have been utilized to construct an expansive collection of nanoarchitectures varying in design, physicochemical properties, cellular processing and biomedical applications. However, the broader therapeutic adaptation of nucleic acid nanoassemblies in general, and RNA-based nanoparticles in particular, have faced several challenges in moving towards (pre)clinical settings. For one, the large-batch synthesis of nucleic acids is still under development, with multi-stranded and chemically modified assemblies requiring greater production capacity while maintaining consistent medical-grade outputs. Furthermore, the unknown immunostimulation by these nanomaterials poses additional challenges, necessary to be overcome for optimizing future development of clinically approved RNA nanoparticles.

Small-Angle Scattering as a Structural Probe for Nucleic Acid Nanoparticles (NANPs) in a Dynamic Solution Environment.

Nucleic acid-based technologies are an emerging research focus area for pharmacological and biological studies because they are biocompatible and can be designed to produce a variety of scaffolds at the nanometer scale. The use of nucleic acids (ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA)) as building materials in programming the assemblies and their further functionalization has recently established a new exciting field of RNA and DNA nanotechnology, which have both already produced a variety of different functional nanostructures and nanodevices. It is evident that the resultant architectures require detailed structural and functional characterization and that a variety of technical approaches must be employed to promote the development of the emerging fields. Small-angle X-ray and neutron scattering (SAS) are structural characterization techniques that are well placed to determine the conformation of nucleic acid nanoparticles (NANPs) under varying solution conditions, thus allowing for the optimization of their design. SAS experiments provide information on the overall shapes and particle dimensions of macromolecules and are ideal for following conformational changes of the molecular ensemble as it behaves in solution. In addition, the inherent differences in the neutron scattering of nucleic acids, lipids, and proteins, as well as the different neutron scattering properties of the isotopes of hydrogen, combined with the ability to uniformly label biological macromolecules with deuterium, allow one to characterize the conformations and relative dispositions of the individual components within an assembly of biomolecules. This article will review the application of SAS methods and provide a summary of their successful utilization in the emerging field of NANP technology to date, as well as share our vision on its use in complementing a broad suite of structural characterization tools with some simulated results that have never been shared before.