RNA Oligonucleotides Research Articles

Electrostatically assembled wound dressings deliver pro-angiogenic anti-miRs preferentially to endothelial cells

Chronic non-healing wounds occur frequently in individuals affected by diabetes, yet standard-of-care treatment leaves many patients inadequately treated or with recurring wounds. MicroRNA (miR) expression is dysregulated in diabetic wounds and drives an anti-angiogenic phenotype, but miRs can be inhibited with short, chemically-modified RNA oligonucleotides (anti-miRs). Clinical translation of anti-miRs is hindered by delivery challenges such as rapid clearance and uptake by off-target cells, requiring repeated injections, excessively large doses, and bolus dosing mismatched to the dynamics of the wound healing process. To address these limitations, we engineered electrostatically assembled wound dressings that locally release anti-miR-92a, as miR-92a is implicated in angiogenesis and wound repair. In vitro, anti-miR-92a released from these dressings was taken up by cells and inhibited its target. An in vivo cellular biodistribution study in murine diabetic wounds revealed that endothelial cells, which play a critical role in angiogenesis, exhibit higher uptake of anti-miR eluted from coated dressings than other cell types involved in the wound healing process. In a proof-of-concept efficacy study in the same wound model, anti-miR targeting anti-angiogenic miR-92a de-repressed target genes, increased gross wound closure, and induced a sex-dependent increase in vascularization. Overall, this proof-of-concept study demonstrates a facile, translational materials approach for modulating gene expression in ulcer endothelial cells to promote angiogenesis and wound healing. Furthermore, we highlight the importance of probing cellular interactions between the drug delivery system and the target cells to drive therapeutic efficacy.

Effect of locoregional injection of RNA oligonucleotide targeting carbohydrate sulfotransferase 15 on both tumoral and lymph node MDSCs and T cell infiltration in pancreatic tumor in mice.

e16310 Background: Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan biosynthetic enzyme and is responsible for tumor matrix remodeling. Intratumoral injection of CHST15 siRNA was shown to increase tumor-infiltrating T cells in patients with pancreatic cancer in the phase I/IIa study. However, the mechanism underlying the enhanced T cell accumulation is not fully explored. Here we investigated the effect of locally injected CHST15 siRNA on modulating tumor immune microenvironment in mice. Methods: For in vivo pharmacokinetics after a single local injection of CHST15 siRNA to normal C57BL/6J mice, RNA oligonucleotides were isolated from tissues and detected by HPLC. Mouse pancreatic cancer KPC cells were implanted subcutaneously into the left hind footpad of syngeneic C57BL/6J mice and the tumor growth was monitored. From week 1, intratumoral injections with 1 mg/mL of CHST15 siRNA or vehicle were performed twice a week for 2 weeks and mice were sacrificed at week 3. In separate experiments, human BxPC-3 cells were implanted subcutaneously in T cell-deficient nude mice, and the effect of intratumoral injections of CHST15 siRNA or vehicle for 2 weeks was also investigated. Immunohistochemical staining was performed for the tumor and tumor-draining lymph node (TDLN). Results: CHST15 siRNA was detectable in not only the local sites but also the draining LNs of normal mice 1 and 4 hours after locoregional injection. In the KPC-implanted syngeneic mouse model, CHST15 siRNA showed significant tumor growth inhibition compared to the vehicle. In tumor, CHST15 was expressed by tumor cells, and to a lesser extent, Ly6C/G+ MDSCs. CHST15 siRNA altered tissue remodeling by repressing dense fibrotic architecture and NETosis. CHST15 siRNA significantly diminished Ly6C/G+ MDSCs while increasing CD4+ and CD8+ T cells. In TDLN, CHST15 was expressed by Ly6C/G+ MDSCs. Locally injected CHST15 siRNA markedly diminished Ly6C/G+ MDSCs, while increasing Ki-67+ proliferating CD4+ and CD8+ T cells. In the BxPC-3-implanted nude mouse model, locally injected CHST15 siRNA repressed excessive fibrosis and NETosis in tumor, diminishing both tumoral and TDLN MDSCs, but tumor growth inhibition was marginal. These results suggest that locally injected CHST15 siRNA could enter into TDLN, directly diminishing CHST15+ MDSCs, and expanded T cells. Tumor growth inhibition was largely T-cell medicated. Conclusions: The present study demonstrated that CHST15 siRNA locally and remotely diminished MDSCs, leading to the enhancement of anti-tumor T cell activity. A novel role of locally-delivered RNA oligonucleotide is suggested.

Light-triggered site-directed RNA editing by endogenous ADAR1 with photolabile guide RNA

RNA A-to-I editing is a post-transcriptional modification pervasively occurring in cells. Artificial intervention of A-to-I editing at specific sites of RNA could also be achieved with guide RNA and exogenous ADAR enzymes. In contrast to previous fused SNAP-ADAR enzymes for light-driven RNA A-to-I editing, we developed photo-caged antisense guide RNA oligonucleotides with simple 3'-terminal cholesterol modification, and successfully achieved light-triggered site-specific RNA A-to-I editing for the first time utilizing endogenous ADAR enzymes. Our caged A-to-I editing system effectively implemented light-dependent point mutation of mRNA transcripts of both exogenous and endogenous genes in living cells and 3D tumorspheres, as well as spatial regulation of EGFP expression, which provides a new approach for precise manipulation of RNA editing.

Novel sights on therapeutic, prognostic, and diagnostics aspects of non-coding RNAs in glioblastoma multiforme.

Glioblastoma Multiforme (GBM) is the primary brain tumor and accounts for 200,000 deaths each year worldwide. The standard therapy includes surgical resection followed by temozolomide (TMZ)-based chemotherapy and radiotherapy. The survival period of GBM patients is only 12-15months. Therefore, novel treatment modalities for GBM treatment are urgently needed. Mounting evidence reveals that non-coding RNAs (ncRNAs) were involved in regulating gene expression, the pathophysiology of GBM, and enhancing therapeutic outcomes. The combinatory use of ncRNAs, chemotherapeutic drugs, and tumor suppressor gene expression induction might provide an innovative, alternative therapeutic approach for managing GBM. Studies have highlighted the role of Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in prognosis and diagnosis. Dysregulation of ncRNAs is observed in virtually all tumor types, including GBMs. Studies have also indicated the blood-brain barrier (BBB) as a crucial factor that hinders chemotherapy. Although several nanoparticle-mediated drug deliveries were degrading effectively against GBM in vitro conditions. However, the potential to cross the BBB and optimum delivery of oligonucleotide RNA into GBM cells in the brain is currently under intense clinical trials. Despite several advances in molecular pathogenesis, GBM remains resistant to chemo and radiotherapy. Targeted therapies have less clinical benefit due to high genetic heterogeneity and activation of alternative pathways. Thus, identifying GBM-specific prognostic pathways, essential genes, and genomic aberrations provide several potential benefits as subtypes of GBM. Also, these approaches will provide insights into new strategies to overcome the heterogenous nature of GBM, which will eventually lead to successful therapeutic interventions toward precision medicine and precision oncology.

A general and efficient approach to synthesize the phosphoramidites of 5′-18 O labeled purine nucleosides

5′-18 O labeled RNA oligos are important probes to investigate the mechanism of 2′-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5′-18 O labeled nucleosides starting from the corresponding commercially available 5′-O-DMT protected nucleosides. Using this method, we prepared 5′-18 O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5′-18 O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5′-18 O-2′-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5′-18 O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2′-O-transphosphorylation reactions.

Advances in Optical Fiber Aptasensor for Biochemical Sensing Applications

AbstractOptical fiber biochemical sensing technology, as a cutting‐edge, multidisciplinary, and universal sensing technology, is gradually becoming the research focus in the field of biochemical sensing. They enjoy distinctive characteristics such as high cost‐effectiveness, flexibility, resistance to electromagnetic interference, and small size and can be used to detect biochemical analytes at ultra‐low concentrations with ultra‐low sample consumption, even when these biochemical analytes are in hard‐to‐reach environments. An aptamer is a DNA or RNA oligonucleotide fragment with a high affinity for the target selected from random sequences. Due to their advantages of simple synthesis, low cost, good stability, good specificity, and strong affinity, they are gradually becoming ideal recognition elements and may replace antibodies as next‐generation target receptors. Therefore, aptamers, as functional materials, are integrated into optical fiber sensors, which are called optical fiber aptasensors, providing new opportunities for the construction of high‐performance and intelligent biochemical sensors. This article aims to review the recent research progress of fiber‐optic aptamer biochemical sensors, with a focus on their sensing mechanisms and applications. Finally, based on the existing deficiencies of fiber‐optic aptamer biochemical sensors and practical requirements, their future development trends are briefly discussed.

Evaluation of Poly(N-Ethyl Pyrrolidine Methacrylamide) (EPA) and Derivatives as Polymeric Vehicles for miRNA Delivery to Neural Cells.

MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells' function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating β-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together.

Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants.

Synthetic RNA oligonucleotides composed of canonical and modified ribonucleotides are highly effective for RNA antisense therapeutics and RNA-based genome engineering applications utilizing CRISPR-Cas9. Yet, synthesis of synthetic RNA using phosphoramidite chemistry is highly inefficient and expensive relative to DNA oligonucleotides, especially for relatively long RNA oligonucleotides. Thus, new biotechnologies are needed to significantly reduce costs, while increasing synthesis rates and yields of synthetic RNA. Here, we engineer human DNA polymerase theta (Polθ) variants and demonstrate their ability to synthesize long (95-200 nt) RNA oligonucleotides with canonical ribonucleotides and ribonucleotide analogs commonly used for stabilizing RNA for therapeutic and genome engineering applications. In contrast to natural promoter-dependent RNA polymerases, Polθ variants synthesize RNA by initiating from DNA or RNA primers, which enables the production of RNA without short abortive byproducts. Remarkably, Polθ variants show the lower capacity to misincorporate ribonucleotides compared to T7 RNA polymerase. Automation of this enzymatic RNA synthesis technology can potentially increase yields while reducing costs of synthetic RNA oligonucleotide production.

The aptamer-based RNA-PROTAC

RNA-binding proteins (RBPs) dysfunction has been implicated in a number of diseases, and RBPs have traditionally been considered to be undruggable targets. Here, targeted degradation of RBPs is achieved based on the aptamer-based RNA-PROTAC, which consists of a genetically encoded RNA scaffold and a synthetic heterobifunctional molecule. The target RBPs can bind to their RNA consensus binding element (RCBE) on the RNA scaffold, while the small molecule can recruit E3 ubiquitin ligase to the RNA scaffold in a non-covalent manner, thereby inducing proximity-dependent ubiquitination and subsequent proteasome-mediated degradation of the target protein. Different RBPs targets, including LIN28A and RBFOX1, have been successfully degraded by simply replacing the RCBE module on the RNA scaffold. In addition, the simultaneous degradation of multiple target proteins has been realized by inserting more functional RNA oligonucleotides into the RNA scaffold.

Middle-out sequence confirmation of CRISPR/Cas9 single guide RNA (sgRNA) using DNA primers and ribonuclease T1 digestion

Accurate sequencing of single guide RNAs (sgRNAs) for CRISPR/Cas9 genome editing is critical for patient safety, as the sgRNA guides the Cas9 nuclease to target site-specific cleavages in DNA. An approach to fully sequence sgRNA using protective DNA primers followed by ribonuclease (RNase) T1 digestion was developed to facilitate the analysis of these larger molecules by hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-HRMS). Without RNase digestion, top-down mass spectrometry alone struggles to properly fragment precursor ions in large RNA oligonucleotides to provide confidence in sequence coverage. With RNase T1 digestion of these larger oligonucleotides, however, bottom-up analysis cannot confirm full sequence coverage due to the presence of short, redundant digestion products. By combining primer protection with RNase T1 digestion, digestion products are large enough to prevent redundancy and small enough to provide base resolution by tandem mass spectrometry to allow for full sgRNA sequence coverage. An investigation into the general requirements for adequate primer protection of specific regions of the RNA was conducted, followed by the development of a generic protection and digestion strategy that may be applied to different sgRNA sequences. This middle-out technique has the potential to expedite accurate sequence confirmation of chemically modified sgRNA oligonucleotides.

Stabilization of the Quadruplex-Forming G-Rich Sequences in the Rhinovirus Genome Inhibits Uncoating-Role of Na+ and K.

Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither vaccines nor other treatments are available. The many existing drug candidates either stabilize the capsid or inhibit the viral RNA polymerase, the viral proteinases, or the functions of other non-structural viral proteins; however, none has been approved by the FDA. Focusing on the genomic RNA as a possible target for antivirals, we asked whether stabilizing RNA secondary structures might inhibit the viral replication cycle. These secondary structures include G-quadruplexes (GQs), which are guanine-rich sequence stretches forming planar guanine tetrads via Hoogsteen base pairing with two or more of them stacking on top of each other; a number of small molecular drug candidates increase the energy required for their unfolding. The propensity of G-quadruplex formation can be predicted with bioinformatics tools and is expressed as a GQ score. Synthetic RNA oligonucleotides derived from the RV-A2 genome with sequences corresponding to the highest and lowest GQ scores indeed exhibited characteristics of GQs. In vivo, the GQ-stabilizing compounds, pyridostatin and PhenDC3, interfered with viral uncoating in Na+ but not in K+-containing phosphate buffers. The thermostability studies and ultrastructural imaging of protein-free viral RNA cores suggest that Na+ keeps the encapsulated genome more open, allowing PDS and PhenDC3 to diffuse into the quasi-crystalline RNA and promote the formation and/or stabilization of GQs; the resulting conformational changes impair RNA unraveling and release from the virion. Preliminary reports have been published.

Aptamer-Based Targeted Protein Degradation.

The selective removal of misfolded, aggregated, or aberrantly overexpressed protein plays an essential role in maintaining protein-dominated biological processes. In parallel, the precise knockout of abnormal proteins is inseparable from the accurate identification of proteins within complex environments. Guided by these precepts, small molecules, or antibodies, are commonly used as protein recognition tools for developing targeted protein degradation (TPD) technology. Indeed, TPD has shown tremendous prospects in chronic diseases, rare diseases, cancer research, and other fields. Meanwhile, aptamers are short RNA or DNA oligonucleotides that can bind to target proteins with high specificity and strong affinity. Accordingly, aptamers are actively used in designing and constructing TPD technology. In this perspective, we provide a brief introduction to TPD technology in its current progress, and we summarize its application challenges. Recent advances in aptamer-based TPD technology are reviewed, together with corresponding challenges and outlooks.

Application of mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles in suppression of laryngeal cancer by targeting cancer stem cells

Although surgery-based comprehensive therapy is becoming the main approach to treat laryngeal cancer, recurrence, metastasis, radiotherapy resistance and chemotherapy tolerance are still the main causes of death in patients. Targeted inhibition of laryngeal cancer stem cells has been considered as the consensus to cure laryngeal cancer. Our previous study has confirmed proto-oncogene Bmi-1 as a key regulator for self-renewal of laryngeal cancer stem cells. Targeted knockdown of Bmi-1 gene effectively inhibited the self-renewal and differentiation of laryngeal cancer stem cells, leading to the promoted sensitivity to chemotherapy including paclitaxel. However, due to off-target effects and quick degradation of the naked Bmi-1-RNAi small RNA oligo by nuclease in body fluids, it is urgently needed to develop a tumor-targeted delivery system with a protective shell. In this study, we designed and synthesized cRGD peptide-modified chitosan-polyethylene glycol slow-release nanoparticles (mPEG-CS-cRGD/Bmi-1RNAi-PTX) containing Bmi-1RNAi siRNA oligo and paclitaxel, which showed spherical in shape, 200 nm diameter in size, low cytotoxicity, strong DNA wrapping, resistance to nuclease degradation and high transfection efficiency to cells. Functional analysis indicated significant suppression of cell proliferation and migration and induction of apoptosis by the nanocomplex in laryngeal cancer cells in vitro. By application to the mouse model with laryngeal cancer, the nanocomplex inhibited tumor growth significantly in vivo. In addition, cRGD peptide, paclitaxel and Bmi-1 siRNA in the nanoparticles showed synergistic effects to suppress laryngeal cancer stem cells. In conclusion, this study not only developed a laryngeal tumor-targeted chemotherapeutic system, but also demonstrated a Bmi-1 RNAi-based chemotherapeutic strategy to inhibit cancer stem cells, having strong potential to treat laryngeal cancer patients suffering therapy resistance and/or tumor recurrence.

RNA Targeting and Gene Editing Strategies for Transthyretin Amyloidosis.

Transthyretin (TTR) is a tetrameric protein synthesized primarily by the liver. TTR can misfold into pathogenic ATTR amyloid fibrils that deposit in the nerves and heart, causing a progressive and debilitating polyneuropathy (PN) and life-threatening cardiomyopathy (CM). Therapeutic strategies, which are aimed at reducing ongoing ATTR amyloid fibrillogenesis, include stabilization of the circulating TTR tetramer or reduction of TTR synthesis. Small interfering RNA (siRNA) or antisense oligonucleotide (ASO) drugs are highly effective at disrupting the complementary mRNA and inhibiting TTR synthesis. Since their development, patisiran (siRNA), vutrisiran (siRNA) and inotersen (ASO) have all been licensed for treatment of ATTR-PN, and early data suggest these drugs may have efficacy in treating ATTR-CM. An ongoing phase 3 clinical trial will evaluate the efficacy of eplontersen (ASO) in the treatment of both ATTR-PN and ATTR-CM, and a recent phase 1 trial demonstrated the safety of novel in vivo CRISPR-Cas9 gene-editing therapy in patients with ATTR amyloidosis. Recent results from trials of gene silencer and gene-editing therapies suggest these novel therapeutic agents have the potential to substantially alter the landscape of treatment for ATTR amyloidosis. Their success has already changed the perception of ATTR amyloidosis from a universally progressive and fatal disease to one that is treatable through availability of highly specific and effective disease-modifying therapies. However, important questions remain including long-term safety of these drugs, potential for off-target gene editing, and how best to monitor the cardiac response to treatment.Kindly check and confirm the processed running title.This is correct.

Increasing the Editing Efficiency of the MS2-ADAR System for Site-Directed RNA Editing

Site-directed RNA editing (SDRE) technologies have great potential in gene therapy. Our group has developed a strategy to redirect exogenous adenosine deaminases acting on RNA (ADARs) to specific sites by making editable structures using antisense RNA oligonucleotides. Improving the editing efficiency of the MS2-ADAR system is important in treating undesirable G-to-A point mutations. This work demonstrates an effective strategy to enhance the editing efficiency of this SDRE system. The strategy involves changing the number of MS2 stem-loops on both sides of the antisense RNA and the mismatch base on the antisense part. The enhanced green fluorescent protein (EGFP) with W58X mutation is used as the reporter gene. Subsequently, we adjusted the amount of plasmids for transfection to tune the expression level of the guide RNA, and finally, we observed the fluorescence signal after transfection. After equalizing number of MS2 stem-loops at both sides of the antisense RNA, high editing efficiency was achieved. In the same level of guide RNA expression, when the paired base position was the target uridine, the editing efficiency was higher than cytidine, adenosine, and guanosine.

DEVELOPMENT OF MURINE STEM CELLS WITH CONDITIONAL KNOCKOUT OF HUMANIZED SNCA GENE

α-synuclein is one of the key molecular links in the pathogenesis of Parkinson’s disease. The accumulated data indicate that pathogenic mutations in the Snca gene are associated with the development of neurodegenerative brain damage, indicating the relevance of studying the synuclein neurobiological role.The aim of the study was to create a genetically modified clone of mouse stem cells with a conditional knockout of humanized α-synuclein, which can be used for the reinjection into mouse blastocysts, as well as for basic and applied in vitro research in the field of pathophysiology and neuropharmacology.Materials and methods. To create mouse stem cells with a conditional knockout of the humanized Snca gene, a previously obtained clone with the first Snca exon flanked by LoxP sites, was used. The CRISPR/Cas9-mediated homologous recombination system with donor DNA oligonucleotides of the human sites of the corresponding gene sites was used to humanize the fourth and fifth exons. Cas9 nuclease, single guide RNA, and donor DNA were transfected into mouse cells.Results. An approach to obtaining clones of mouse genetically modified stem cells expressing pathological humanized α-synuclein, has been proposed and implemented. The resulting clones were plated on Petri dishes for propagation and a further genetic analysis. Clone 126-2F4 was found out carrying the necessary genetic modifications. The results obtained are fundamentally important not only for understanding the development of the pathological process in α-synucleinopathies, but which is more important, for the development of new therapeutic approaches that will stop the extension of the human α-synuclein aggregation pathology throughout the nervous system, and the validation of these approaches in preclinical trials.Conclusion. As a result of the study, a strategy for CRISPR/Cas9-assisted homologous recombination in the genome of mouse embryonic stem cells has been developed to create a fully humanized Snca gene encoding α-synuclein, and the clone genome of mouse embryonic stem cells has been edited using a CRISPR technology. The RNA and DNA oligonucleotides necessary for the creation of RNP complexes that carry out a directed homologous recombination in the Snca locus of the mouse genome have been synthesized. The developed cell clone can serve to create a line of genetically modified mice that serve as a test system for pathophysiological and neuropharmacological studies associated with synucleinopathies. Herewith, before the induction of the Cre-dependent recombination, this line is a representative model for studying a biological role of mutant Snca. At the same time, after a Cre-dependent knockout activation, it is possible to imitate the pharmacological inhibition of α-synuclein, which is of particular interest for applied research in neuropharmacology.

Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2.

Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein-RNA and protein-DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5'-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization.

MicroRNA-210 Downregulates TET2 (Ten-Eleven Translocation Methylcytosine Dioxygenase 2) and Contributes to Neuroinflammation in Ischemic Stroke of Adult Mice.

Stroke is a leading cause of morbidity and mortality worldwide. Neuroinflammation plays a key role in acute brain injury of ischemic stroke. MicroRNA-210 (miR210) is the master hypoxamir and regulates microglial activation and inflammation in a variety of diseases. In this study, we uncovered the mechanism of miR210 in orchestrating ischemic stroke-induced neuroinflammation through repression of TET2 (ten-eleven translocation methylcytosine dioxygenase 2) in the adult mouse brain. Ischemic stroke was induced in adult WT (wild type) or miR210 KO (miR210 deficient) mice by transient intraluminal middle cerebral artery occlusion. Injection of TET2 silencing RNA or miR210 complementary locked nucleic acid oligonucleotides, or miR210 KO mice were used to validate miR210-TET2 axis and its role in ischemic brain injury. Furthermore, the effect of TET2 overexpression on miR210-stimulated proinflammatory cytokines was examined in BV2 microglia. Post assays included magnetic resonance imaging scan for brain infarct size; neurobehavioral tests, reverse transcription-quantitative polymerase chain reaction, and Western blot for miR210; and TET2 levels, flow cytometry, and ELISA for neuroinflammation in the brain after stroke or microglia in vitro. miR210 injection significantly reduced TET2 protein abundance in the brain, while miR210 complementary locked nucleic acid oligonucleotides or miR210 KO preserved TET2 regardless of ischemic brain injury. TET2 knockdown reversed the protective effects of miR210 inhibition or miR210 KO on ischemic stroke-induced brain infarct size and neurobehavioral deficits. Moreover, flow cytometry and ELISA assays showed that TET2 knockdown also significantly dampened the anti-inflammatory effect of miR210 inhibition on microglial activation and IL (interleukin)-6 release after stroke. In addition, overexpression of TET2 in BV2 microglia counteracted miR210-induced increase in cytokines. miR210 inhibition reduced ischemic stroke-induced neuroinflammatory response via repression of TET2 in the adult mouse brain, suggesting that miR210 is a potential treatment target for acute brain injury after ischemic stroke.

Preparation of a Convertible Spacer Containing a Disulfide Group for Versatile Functionalization of Oligonucleotides

The protocols described in this article provide details regarding the synthesis and characterization of a disulfide containing linker phosphoramidite for terminal functionalization of synthetic oligonucleotides. The linker is first synthesized from 6-mercaptohexanol in two steps and is incorporated at the 5' end of short DNA oligonucleotides using automated solid-phase synthesis. The linker contains a terminal tosylate group which is post-synthetically displaced by altering the deprotection conditions to yield a variety of functional handles (N3 , NH2 , OMe, SH) or alternatively, the tosylate can be displaced directly with primary amines such as tert-butylamine. The linker system is also compatible with RNA oligonucleotides enabling the introduction of various functional handles (N3 , NH2 ). The protocol outlined in this procedure provides access to a versatile linker for the terminal functionalization of oligonucleotides containing a disulfide bond which may serve useful in the synthesis of reduction-responsive oligonucleotide conjugates. As a proof of concept, in this protocol the linker is used to modify a dT10 oligonucleotide and then conjugated by copper(I)-mediated azide-alkyne cycloaddition (CuAAC) to an alkyne-modified poly(ethylene glycol) which shows concentration dependent release of the oligonucleotide upon treatment with 1,4-dithiothreitol, a reducing agent. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of disulfide linker phosphoramidite 3 Basic Protocol 2: Synthesis, functionalization, and characterization of DNA oligonucleotides containing disulfide linker phosphoramidite 3 Basic Protocol 3: Displacement of terminal tosylate functionalized DNA with primary aliphatic amines Basic Protocol 4: Synthesis of oligonucleotide-PEG conjugate Support Protocol: Preparation of PEG-alkyne.

Tuning Material States and Functionalities of G-Quadruplex-Modulated RNA-Peptide Condensates

RNA encodes sequence- and structure-dependent interactions to modulate the assembly and properties of biomolecular condensates. RNA G-quadruplexes (rG4s) formed by guanine-rich sequences can trigger the formation of liquid- or solid-like condensates that are involved in many aberrant phase transitions. However, exactly how rG4 motifs modulate different phase transitions and impart distinct material properties to condensates is unclear. Here, using RNA oligonucleotides and cationic peptides as model systems, we show that RNA-peptide condensates exhibit tunability in material properties over a wide spectrum via interactions arising from rG4 folding/unfolding kinetics. rG4-containing oligonucleotides formed strong pairwise attraction with peptides and tended to form solid-like condensates, while their less-structured non-G4 mutants formed liquid-like droplets. We find that the coupling between rG4 dissociation and RNA-peptide complex coacervation triggers solid-to-liquid transition of condensates prior to the complete unfolding of rG4s. This coupling points to a mechanism that material states of rG4-modulated condensates can be finely tuned from solid-like to liquid-like by the addition of less-structured RNA oligonucleotides, which have weak but dominant binding with peptides. We further show that the tunable material states of condensates can enhance RNA aptamer compartmentalization and RNA cleavage reactions. Our results suggest that condensates with complex properties can emerge from subtle changes in RNA oligonucleotides, contributing ways to treat dysfunctional condensates in diseases and insights into prebiotic compartmentalization.