Functionally-interdependent shape-switching nanoparticles with controllable properties.

We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

Programmable Nucleic Acid Based Polygons with Controlled Neuroimmunomodulatory Properties for Predictive QSAR Modeling.

In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential to understand the immunostimulatory aptitude of such tools and identify potential complications. This paper presents a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. It is demonstrated that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance the current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, quantitative structure-activity relationship modeling is conducted. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predicts the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.

Versatile RNA tetra-U helix linking motif as a toolkit for nucleic acid nanotechnology.

RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community.

RNA methyltransferase METTL16: Targets and function.

The N6-methyladenosine (m6A) RNA methyltransferase METTL16 is an emerging player in the RNA modification landscape of the human cell. Originally thought to be a ribosomal RNA methyltransferase, it has now been shown to bind and methylate the MAT2A messenger RNA (mRNA) and U6 small nuclear RNA (snRNA). It has also been shown to bind the MALAT1 long noncoding RNA and several other RNAs. METTL16's methyltransferase domain contains the Rossmann-like fold of class I methyltransferases and uses S-adenosylmethionine (SAM) as the methyl donor. It has an RNA methylation consensus sequence of UACAGARAA (modified A underlined), and structural requirements for its known RNA interactors. In addition to the methyltransferase domain, METTL16 protein has two other RNA binding domains, one of which resides in a vertebrate conserved region, and a putative nuclear localization signal. The role of METTL16 in the cell is still being explored, however evidence suggests it is essential for most cells. This is currently hypothesized to be due to its role in regulating the splicing of MAT2A mRNA in response to cellular SAM levels. However, one of the more pressing questions remaining is what role METTL16's methylation of U6 snRNA plays in splicing and potentially cellular survival. METTL16 also has several other putative coding and noncoding RNA interactors but the definitive methylation status of those RNAs and the role METTL16 plays in their life cycle is yet to be determined. Overall, METTL16 is an intriguing RNA binding protein and methyltransferase whose important functions in the cell are just beginning to be understood. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Building Interdisciplinary Partnerships for Community-Engaged Environmental Health Research in Appalachian Virginia.

This article describes a collaboration among a group of university faculty, undergraduate students, local governments, local residents, and U.S. Army staff to address long-standing concerns about the environmental health effects of an Army ammunition plant. The authors describe community-responsive scientific pilot studies that examined potential environmental contamination and a related undergraduate research course that documented residents' concerns, contextualized those concerns, and developed recommendations. We make a case for the value of resource-intensive university-community partnerships that promote the production of knowledge through collaborations across disciplinary paradigms (natural/physical sciences, social sciences, health sciences, and humanities) in response to questions raised by local residents. Our experience also suggests that enacting this type of research through a university class may help promote researchers' adoption of "epistemological pluralism", and thereby facilitate the movement of a study from being "multidisciplinary" to "transdisciplinary".

Characterization of METTL16 as a cytoplasmic RNA binding protein.

mRNA modification by N6-methyladenosine (m6A) is involved in many post-transcriptional regulation processes including mRNA stability, splicing and promotion of translation. Accordingly, the recently identified mRNA methylation complex containing METTL3, METTL14, and WTAP has been the subject of intense study. However, METTL16 (METT10D) has also been identified as an RNA m6A methyltransferase that can methylate both coding and noncoding RNAs, but its biological role remains unclear. While global studies have identified many potential RNA targets of METTL16, only a handful, including the long noncoding RNA MALAT1, the snRNA U6, as well as the mRNA MAT2A have been verified and/or studied to any great extent. In this study we identified/verified METTL16 targets by immunoprecipitation of both endogenous as well as exogenous FLAG-tagged protein. Interestingly, exogenously overexpressed METTL16 differed from the endogenous protein in its relative affinity for RNA targets which prompted us to investigate METTL16's localization within the cell. Surprisingly, biochemical fractionation revealed that a majority of METTL16 protein resides in the cytoplasm of a number of cells. Furthermore, siRNA knockdown of METTL16 resulted in expression changes of a few mRNA targets suggesting that METTL16 may play a role in regulating gene expression. Thus, while METTL16 has been reported to be a nuclear protein, our findings suggest that METTL16 is also a cytoplasmic methyltransferase that may alter its RNA binding preferences depending on its cellular localization. Future studies will seek to confirm differences between cytoplasmic and nuclear RNA targets in addition to exploring the physiological role of METTL16 through long-term knockdown.

Substance Use in Rural Central Appalachia: Current Status and Treatment Considerations.

The burden of substance use and especially the unmatched rates of overdoses in rural Central Appalachia highlight the need for innovative approaches to curb the initiation to drug misuse and to address current substance use disorders. Effective substance use interventions involve a thorough understanding of the region. In Central Appalachia, many of the barriers to treatment are shared with other rural and impoverished areas, including a lack of access to health care and lack of health care providers with specialized training. Parts of Appalachia also present their own considerations, including the challenges of fostering trust and encouraging treatment-seeking in communities with dense, long-term, place-based social and family networks. Current policies and interventions for substance use have been largely inadequate in the region, as evidenced by continued increases in substance use and substance-related deaths, especially related to nonmedical prescription drug use and increasing heroin use. The authors discuss ways in which rural life, poverty, identity, and values in Appalachia have influenced substance use and treatment and propose strategies and interventions to improve outcomes.

Environmental health disparities in the Central Appalachian region of the United States.

Health disparities that cannot be fully explained by socio-behavioral factors persist in the Central Appalachian region of the United States. A review of available studies of environmental impacts on Appalachian health and analysis of recent public data indicates that while disparities exist, most studies of local environmental quality focus on the preservation of nonhuman biodiversity rather than on effects on human health. The limited public health studies available focus primarily on the impacts of coal mining and do not measure personal exposure, constraining the ability to identify causal relationships between environmental conditions and public health. Future efforts must engage community members in examining all potential sources of environmental health disparities to identify effective potential interventions.

Triggerable RNA nanodevices.

The targeted and conditional activation of pharmaceuticals is an increasingly important feature in modern personalized medicine. Nucleic acid nanoparticles show tremendous potential in this exploit due to their programmability and biocompatibility. Among the most powerful nucleic acid specific treatments is RNA interference-based therapeutics. RNA interference is a naturally occurring phenomenon in which specific genes are effectively silenced. Recently we have developed two different strategies based on customized multivalent nucleic acid nanoparticles with the ability to conditionally activate RNA interference in diseased cells as well as elicit detectable fluorescent responses.[1,2] These novel technologies can be further utilized for the simultaneous delivery and conditional intracellular activation of multiple therapeutic and biosensing functions to combat various diseases.

Cellular Delivery of RNA Nanoparticles.

RNA nanostructures can be programmed to exhibit defined sizes, shapes and stoichiometries from naturally occurring or de novo designed RNA motifs. These constructs can be used as scaffolds to attach functional moieties, such as ligand binding motifs or gene expression regulators, for nanobiology applications. This review is focused on four areas of importance to RNA nanotechnology: the types of RNAs of particular interest for nanobiology, the assembly of RNA nanoconstructs, the challenges of cellular delivery of RNAs in vivo, and the delivery carriers that aid in the matter. The available strategies for the design of nucleic acid nanostructures, as well as for formulation of their carriers, make RNA nanotechnology an important tool in both basic research and applied biomedical science.