In Vivo Study of HIV-1 Tat Arginine-rich Motif Unveils Its Transport Properties

Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery as they incorporate specific attributes required for efficient cargo delivery to sub-cellular domains. Little is known, however, about intracellular trafficking and interactions of Tat peptide–tagged cargoes, although some in vitro studies have suggested the relevance of active processes in Tat peptide–driven nuclear translocation. These issues are addressed by comparing Tat peptide–induced transport properties with well-established passive diffusion and active import benchmarks in living cells. Specifically, we examine several constructs of increasing molecular weight (MW) both below and above the threshold for passive diffusion through the nuclear pore. The resulting sub-cellular localization is analyzed by confocal imaging, and construct intracellular dynamics is investigated by fluorescence recovery after photobleaching (FRAP) real-time imaging. Our experiments yield the characteristic transport parameters of Tat peptide intra-cytoplasm dynamics and nucleus/cytoplasm shuttling. These results allow us to elucidate the mechanism of Tat peptide–driven nuclear permeation, demonstrating that it crosses the nuclear envelope (NE) by passive diffusion. Finally, we discuss the limitations of this route in terms of acceptable cargo size

Live-cell imaging of non-coding RNAs dynamics in ALS condensates

In the last few years, interest around non-coding RNAs (ncRNAs) has been growing as they have been found to be involved in several physiological and pathological processes. In fact, their expression is highly enriched in neuronal tissues and, thanks to their complex and modular secondary structure, they can work as scaffold for other RNAs and proteins for the assembly of ribonucleoparticles (RNPs). These supramolecular structures are known to participate in axonal trafficking, a process usually impaired in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and in particular in the presence of mutations of several RNA binding proteins, among which FUS. In this context, it is crucial to investigate ncRNA dynamics and kinetics in live cells, in order to unveil novel mechanisms for the understanding of neurodegeneration. For this purpose, we managed to engineer the motor neuron enriched circRNA circ-Hdgfrp3 and the lncRNA HOTAIRM1 with an array of Pepper, a novel fluorescent aptamer that shows enhanced stability and brightness if compared with previously described fluorescent RNAs, allowing robust RNA imaging with minimal target perturbation. Combining widefield and structured illumination microscopy, we were able to confirm in live mammalian cells that circ-Hdgfrp3 is loaded in G3BP1 and FUSmut RNPs, possibly determining the mechanism through which it is recruited in pathological aggregates in motor neurons. Moreover, we also observed its interaction with DCP1A-tagged processing bodies, raising promising insight about its function and metabolism. Notably, we also determined HOTAIRM1 constitutive participation in stress granules, while we observed its involvement in the dynamics of FUSmut aggregation. Moreover, we were able to follow its behavior throughout the induction of oxidative stress, an event that leads to the production of aggregates containing several RNA binding proteins, including FUSmut. Via live imaging assays, we determined that its recruitment in stress granules is not mediated neither by G3BP1 nor by FUS, consistent with the observation that it does not interfere with stress granules assembly and that it preferentially joins the outer layers of such structures. Overall, as fluorescent RNA technologies are rapidly spreading and are improved for live-imaging applications, our work provides a novel approach for the investigation of ncRNAs’ implication in neurodegenerative diseases with a super-resolution potential in live neuronal cells

RNA localization in neurite morphogenesis and synaptic regulation: current evidence and novel approaches

It is now generally accepted that RNA localization in the central nervous system conveys important roles both during development and in the adult brain. Of special interest is protein synthesis located at the synapse, as this potentially confers selective synaptic modification and has been implicated in the establishment of memories. However, the underlying molecular events are largely unknown. In this review, we will first discuss novel findings that highlight the role of RNA localization in neurons. We will focus on the role of RNA localization in neurotrophin signaling, axon outgrowth, dendrite and dendritic spine morphogenesis as well as in synaptic plasticity. Second, we will briefly present recent work on the role of microRNAs in translational control in dendrites and its implications for learning and memory. Finally, we discuss recent approaches to visualize RNAs in living cells and their employment for studying RNA trafficking in neurons

Antisense Silver Nanoparticles for Photo-activated Gene Silencing

The unique properties of noble metal nanoparticles, which include tunable electronic and photonic characteristics, contribute to their potential as novel delivery vectors with enhanced drug stability, cell uptake, and photo-activated functionalities. Silver, as one of best surface-enhancing substrates available for bulk nanostructure synthesis, is a prime choice for investigations of metal nanohybrids as antisense therapy vehicles with special surface plasmon resonance (SPR) enabled functional attributes. The singular photonic properties of silver nanoparticles (SNPs) may contribute to ease of delivery confirmation and in situ photo-activation of protected cargo packed on particle surfaces. Here we show the synthesis and characterization of 40-80nm SNPs designed for enhanced antisense oligonucleotide delivery and photo-activated gene silencing. Non-active (caged) SNP-bound DNA oligonucleotides possess an internal nitrobenzyl photocleavable linker which once exposed to light, initiates disengagement of functional antisense oligonucleotides from the nanohybrid surface. We demonstrate light-triggered, spatiotemporally controlled gene silencing based on SNP-antisense conjugates, which prove to be promising alternative platforms for gene therapy, gene expression studies, and other nanomedicine applications

Deciphering the Details of RNA Aminoglycoside Interactions: From Atomistic Models to Biotechnological Applications

Aminoglycosides are a class of antibiotics functioning through binding to 16S rRNA A-site and inhibiting the bacterial translation. However, the continuous emergence of drug-resistant strains makes the development of new and more potent antibiotics necessary. Aminoglycosides are also known to interact with various biologically crucial RNA molecules other than 16S rRNA A-site and inhibit their functions. As a result, they are considered as the single most important model to understand the principles of RNA small molecule recognition. The detailed understanding of these interactions is necessary for the development of novel antibacterial, antiviral or even anti-oncogenic agents. In our studies, we have studied both the natural aminoglycoside targets like Rev responsive element (RRE), trans-activating region (TAR) of HIV-1 and thymidylate synthase mRNA 5\u27 untranslated (UTR) region as well as the in vitro selected neomycin, tobramycin and kanamycin RNA aptamers. By this way, we think we have covered a variety of binding pockets to figure out the critical nucleic acid residues playing essential role in aminoglycoside recognition. Along with all these RNAs, we studied more than 10 aminoglycoside ligands to pinpoint the chemical groups in close contact with RNAs. To determine thermodynamic parameters for these interactions, we utilized isothermal titration calorimetry (ITC) assay by which we found that the majority of these interactions are enthalpy driven. More specifically, RNA aminoglycoside interactions are mainly derived by electrostatic and hydrogen binding interactions. Our studies indicated that the amino groups on the first ring of the aminoglycosides are essential for high affinity binding whereas having bulky groups on ring II sterically eliminate their interactions with RNAs. RNA binding trend of aminoglycosides are as follows: neomycin-B \u3e ribostamycin \u3e kanamycin-B \u3e tobramycin \u3e paromomycin \u3e sisomicin \u3e gentamicin \u3e kanamycin-A \u3e geneticin \u3e amikacin \u3e netilmicin. Aminoglycoside binding to the aptamer was shown highly buffer dependent. This phenomenon was analyzed in five different buffers and found that cacodylate-based buffer changes the specificity of the aptamer. In addition to ITC, we have used molecular docking to specifically find out the chemical groups in these interactions. We have specified the nucleic acid residues interacting with aminoglycosides. In parallel, molecular dynamics (MD) simulations of neomycin RNA aptamer with neomycin-B in an all-atom platform in GROMACS were carried out. The results showed a mobile structure consistent with the ability of this aptamer to interact with a wide range of ligands. From molecular docking and MD simulations, we identified the neomycin-B aptamer residues that might contribute to its ligand selectivity and designed a series of new aptamers accordingly. Also, A16 was found to be flexible, which was confirmed by 2AP fluorescence studies. In this analysis, the buffer dependence was also confirmed against neomycin-B, ribostamycin and paromomycin. One of the challenges in therapeutics is the emergence of resistant cells. They become reistant to the drugs via changing the target site, or enzymatically modifying the drug, or producing drug pumps to export the drugs. To overcome the very last challenge, we are utilizing RNA-aminoglycoside partners to keep high intracellular drug concentration and increase the efficacy of aminoglycosides against bacteria. We called the system as DRAGINs (Drug binding aptamers for growing intracellular numbers). We express these RNAs in bacteria and detect their growth rate in order to evaluate their response to different concentration of aminoglycosides. In this study, we found that we could successfully decrease the IC50 values by 2 to 5 fold with the help of aminoglycoside-binding RNA aptamers. Finally, we are mathematically modeling the effect of aptamers on IC50 values of drugs with the use of four-compartment model. In our research group, we are utilizing these RNA-aminoglycoside partners to develop tags for detecting RNA in vivo and in real time. We called this system as intracellular multiaptamer genetic tags (IMAGEtags)

Nucleic Acid Architectures for Therapeutics, Diagnostics, Devices and Materials

Nucleic acids (RNA and DNA) and their chemical analogs have been utilized as building materials due to their biocompatibility and programmability. RNA, which naturally possesses a wide range of different functions, is now being widely investigated for its role as a responsive biomaterial which dynamically reacts to changes in the surrounding environment. It is now evident that artificially designed self-assembling RNAs, that can form programmable nanoparticles and supra-assemblies, will play an increasingly important part in a diverse range of applications, such as macromolecular therapies, drug delivery systems, biosensing, tissue engineering, programmable scaffolds for material organization, logic gates, and soft actuators, to name but a few. The current exciting Special Issue comprises research highlights, short communications, research articles, and reviews that all bring together the leading scientists who are exploring a wide range of the fundamental properties of RNA and DNA nanoassemblies suitable for biomedical applications

Fluorescent Labeling, Co-Tracking, and Quantification of RNA In Cellulo.

RNA plays a fundamental, pervasive role in cellular physiology, through the maintenance and controlled readout of all genetic information, a functional landscape we are only beginning to understand. In particular, the cellular mechanisms for the spatiotemporal control of the plethora of RNAs are still poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent biophysical and biochemical parameters that govern cellular RNA functions, including those of protein-encoding mRNAs. Yet progress has been hampered by the scarcity of high-yield, efficient methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ a series of in vitro enzymatic techniques to efficiently, extensively and in high-yield, incorporate chemically modified nucleoside triphosphates into a transcribed messenger RNA body, between its body and tail (BBT), or randomly throughout the poly(A) tail (tail). Of these, BBT and tail modified strategies proved the most promising methods to functionally label messenger RNA and single-particle track their behaviors using our in-house single-molecule assay: intracellular single-molecule high resolution localization and counting (iSHiRLoC). From this research also was spawned a novel method to anchor an RNA to the actin cytoskeleton for the study of long-term interactions within a cellular context, termed: Gene-Actin Tethered Intracellular Co-tracking Assay (GATICA). Here, biotinylated RNA is tethered to the actin surface, either through complexation with a streptavidin coupled to a biotinylated phalloidin molecule or actin protein. Taken together, this body of work represents strategies for the labeling and visualizing, both freely diffusing and actin tethered, long-RNAs and their interactome in real-time.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135832/1/tcuster_1.pd