Binding of RNA Aptamers to Membrane Lipid Rafts: Implications for Exosomal miRNAs Transfer from Cancer to Immune Cells

Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP–RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.     

The Role of Exosomal Non-Coding RNAs in Colorectal Cancer Drug Resistance

Background: Colorectal cancer (CRC) is one of the most common types of cancer diagnosed worldwide with high morbidity; drug resistance is often responsible for treatment failure in CRC. Non-coding RNAs (ncRNAs) play distinct regulatory roles in tumorigenesis, cancer progression and chemoresistance. Methods: A literature search was conducted in PubMed database in order to sum up and discuss the role of exosomal ncRNAs (ex-ncRNAs) in CRC drug resistance/response and their possible mechanisms. Results: Thirty-six (36) original research articles were identified; these included exosome or extracellular vesicle (EV)-containing microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and small-interfering (siRNAs). No studies were found for piwi-interacting RNAs. Conclusions: Exosomal transfer of ncRNAs has been documented as a new mechanism of CRC drug resistance. Despite being in its infancy, it has emerged as a promising field for research in order to (i) discover novel biomarkers for therapy monitoring and/or (ii) reverse drug desensitization.     

Oncogenic Role of Exosomal Circular and Long Noncoding RNAs in Gastrointestinal Cancers

Circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) are differentially expressed in gastrointestinal cancers. These noncoding RNAs (ncRNAs) regulate a variety of cellular activities by physically interacting with microRNAs and proteins and altering their activity. It has also been suggested that exosomes encapsulate circRNAs and lncRNAs in cancer cells. Exosomes are then discharged into the extracellular environment, where they are taken up by other cells. As a result, exosomal ncRNA cargo is critical for cell–cell communication within the cancer microenvironment. Exosomal ncRNAs can regulate a range of events, such as angiogenesis, metastasis, immune evasion, drug resistance, and epithelial-to-mesenchymal transition. To set the groundwork for developing novel therapeutic strategies against gastrointestinal malignancies, a thorough understanding of circRNAs and lncRNAs is required. In this review, we discuss the function and intrinsic features of oncogenic circRNAs and lncRNAs that are enriched within exosomes.     

Spotlight on Exosomal Non-Coding RNAs in Breast Cancer: An In Silico Analysis to Identify Potential lncRNA/circRNA-miRNA-Target Axis

Breast cancer (BC) has recently become the most common cancer type worldwide, with metastatic disease being the main reason for disease mortality. This has brought about strategies for early detection, especially the utilization of minimally invasive biomarkers found in various bodily fluids. Exosomes have been proposed as novel extracellular vesicles, readily detectable in bodily fluids, secreted from BC-cells or BC-tumor microenvironment cells, and capable of conferring cellular signals over long distances via various cargo molecules. This cargo is composed of different biomolecules, among which are the novel non-coding genome products, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and the recently discovered circular RNA (circRNA), all of which were found to be implicated in BC pathology. In this review, the diverse roles of the ncRNA cargo of BC-derived exosomes will be discussed, shedding light on their primarily oncogenic and additionally tumor suppressor roles at different levels of BC tumor progression, and drug sensitivity/resistance, along with presenting their diagnostic, prognostic, and predictive biomarker potential. Finally, benefiting from the miRNA sponging mechanism of action of lncRNAs and circRNAs, we established an experimentally validated breast cancer exosomal non-coding RNAs-regulated target gene axis from already published exosomal ncRNAs in BC. The resulting genes, pathways, gene ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis could be a starting point to better understand BC and may pave the way for the development of novel diagnostic and prognostic biomarkers and therapeutics.     

Graph Theoretical Methods and Workflows for Searching and Annotation of RNA Tertiary Base Motifs and Substructures

The increasing number and complexity of structures containing RNA chains in the Protein Data Bank (PDB) have led to the need for automated structure annotation methods to replace or complement expert visual curation. This is especially true when searching for tertiary base motifs and substructures. Such base arrangements and motifs have diverse roles that range from contributions to structural stability to more direct involvement in the molecule’s functions, such as the sites for ligand binding and catalytic activity. We review the utility of computational approaches in annotating RNA tertiary base motifs in a dataset of PDB structures, particularly the use of graph theoretical algorithms that can search for such base motifs and annotate them or find and annotate clusters of hydrogen-bond-connected bases. We also demonstrate how such graph theoretical algorithms can be integrated into a workflow that allows for functional analysis and comparisons of base arrangements and sub-structures, such as those involved in ligand binding. The capacity to carry out such automatic curations has led to the discovery of novel motifs and can give new context to known motifs as well as enable the rapid compilation of RNA 3D motifs into a database.     

异丙酚与支撑双层类脂膜作用的电化学研究

以支撑双层类脂膜(s-BLM)作为生物膜模型,采用循环伏安法和交流阻抗技术研究了异丙酚与双层类脂膜的相互作用.结果表明,异丙酚能降低磷脂分子的有序性, 诱发s-BLM 上形成孔洞或缺陷, 且这种相互作用对作用时间、异丙酚浓度以及胆固醇的存在与否具有依赖性.     

Strategy for Internal Labeling of Large RNAs with Minimal Perturbation by Using Fluorescent PNA

Fluorescence techniques for the investigation of biomolecules and their folding pathways require an efficient labeling strategy. A common method to internally label large RNAs involves the introduction of long loops for hybridization of fluorophore‐carrying DNA strands. Such loops often disturb the structure, and thus the functionality, of the RNA. Here we show, in a proof of concept study with a >600 nucleotide group II intron ribozyme, that the usage of the nucleic acid analogue peptide nucleic acid (PNA) is more efficient in several aspects, minimizing the required structural modifications of the RNA. We demonstrate by various methods, including smFRET, that much smaller concentrations and shorter PNAs can be applied, compared to DNA, for rapid and specific internal RNA labeling. The folding pathway and catalytic activity of this large ribozyme is only minimally affected by the PNA, but the background signal is significantly reduced.     

High‐Fidelity Recognition of RNA: Solution Structure of a DNA:RNA Hybrid Duplex with a Molecular Cap

Binding RNA targets, such as microRNAs, with high fidelity is challenging, particularly when the nucleobases to be bound are located at the terminus of the duplex between probe and target. Recently, a peptidyl chain terminating in a quinolone, called ogOA, was shown to act as a cap that enhances affinity and fidelity for RNAs, stabilizing duplexes with Watson–Crick pairing at their termini. Here we report the three‐dimensional structure of an intramolecular complex between a DNA strand featuring the ogOA cap and an RNA segment, solved by NMR and restrained torsion angle molecular dynamics. The quinolone stacks on the terminal base pair of the hybrid duplex, positioned by the peptidyl chain, whose prolinol residue induces a sharp bend between the 5′ terminus of the DNA chain and the glycine linked to the oxolinic acid residue. The structure explains why canonical base pairing is favored over hard‐to‐suppress mismatched base combinations, such as T:G and A:A, and helps to design improved high‐fidelity probes for RNA.     

Strategies and Perspectives for UV Resonance Raman Applicability in Clinical Analyses of Human Sperm RNA

Developing a deeper knowledge about the impact of DNA and RNA epigenetic mutations on sperm production and fertilization performance is essential for selecting best quality samples in Assisted Reproductive Technologies (ART). Indeed, sperm RNAs adenine and guanine are likely to be methylated in low quality RNA sperm samples and their study requires the employment of techniques able to isolate high quality nucleic acids. UV resonance Raman spectroscopy represents a valuable tool that is able to monitor peculiar molecular modifications occurring predominantly in nucleic acids, being less sensitive to the presence of other biological compounds. In this work, we used an UV Resonance Raman (UVRR) setup coupled to a synchrotron radiation source tuned at 250 nm, in order to enhance sperm RNAs adenine and guanine vibrational signals, reducing also the impact of a fluorescence background typically occurring at lower energies. Despite that our protocol should be further optimized and further analyses are requested, our results support the concept that UVRR can be applied for setting inexpensive tools to be employed for semen quality assessment in ART.     

Droplet‐Shooting and Size‐Filtration (DSSF) Method for Synthesis of Cell‐Sized Liposomes with Controlled Lipid Compositions

We report a centrifugal microfluidic method, droplet‐shooting and size‐filtration (DSSF), for the production of cell‐sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil‐water interface, thus resulting in the generation of cell‐sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5–2 μL) of sample solution is required for the formation of hundreds of cell‐sized liposomes. We believe that this method can be applied to generate cell‐sized liposomes for a wide variety of uses, such as the construction of artificial cell‐like systems.