Uncovering the Physical Basis for Selective Mixing Versus Unmixing of Protein-RNA Condensates
0
Citation
0
Reference
10
Related Paper
Among the many different types of molecules that form clathrate hydrates, H2 is unique as it can easily diffuse into and out of clathrate cages, a process that involves the physical–chemical interactions between guest (H2) and host (water) molecules, and is unlike any other molecular system. The dynamic and nano-scale process of H2 diffusion into binary structure II hydrates, where the large cages are occupied by larger molecules, was studied using molecular dynamics simulation. As the H2 molecules diffused from one cage to another, two types of diffusion processes were observed: (i) when moving between a pair of large cages, the H2 molecules pass through the central part of the hexagonal rings; (ii) however, when the H2 molecules move from a large cage to a small one, it requires one of the pentagonal rings to partially break, as this allows the H2 molecule to pass through the widened space. While the diffusion of H2 molecules between large cages was found to occur more frequently, the presence of SF6 molecules in the large cages was found to inhibit diffusion. Therefore, in order to attain higher H2 storage capacities in binary hydrates, it is suggested that there is an optimal number of large cages that should be occupied by SF6 molecules.
Clathrate hydrate
Molecular diffusion
Cite
Citations (13)
Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS.We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq(®) RNA Access Library Preparation Kit.Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.
Cite
Citations (48)
Nuclease protection assay
Strain (injury)
Cite
Citations (96)
Abstract Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom‐up fabrication of nanodevices. Unlike DNA, RNA is a more versatile molecule whose range in the cell is from 21 to thousands of nucleotides and is usually folded into stem and loop structures. RNA is unique in nanoscale fabrication due to its diversity in size, function, and structure. Because gene expression analysis is becoming a clinical reality and there is a need to collect RNA in minute amounts from clinical samples, keeping the RNA intact is a growing challenge. RNA samples are notoriously difficult to handle because of their highly labile nature and tendency to degrade even under controlled RNase‐free conditions and maintenance in the cold. Silencing the RNA that induces the RNA interference is viewed as the next generation of therapeutics. The stabilization and delivery of RNA to cells are the major concerns in making siRNAs usable drugs. For the first time, ultrasonic waves are shown to convert native RNA molecules to RNA nanospheres. The creation of the nanobubbles is performed by a one‐step reaction. The RNA nanospheres are stable at room temperature for at least one month. Additionally, the nanospheres can be inserted into mammalian cancer cells (U2OS). This research achieves: 1) a solution to RNA storage; and 2) a way to convert RNA molecules to RNA particles. RNA nanosphere formation is a reversible process, and by using denaturing conditions, the RNA can be refolded into intact molecules.
Nuclease protection assay
RNA Silencing
Cite
Citations (38)
DNA and RNA can spontaneously self-assemble into various structures, including aggregates, complexes, and ordered structures. The self-assembly reactions cannot be genetically encoded to occur in living mammalian cells since the double-stranded nucleic acids generated by current self-assembly approaches are unstable and activate innate RNA immunity pathways. Here, we show that recently described dimeric aptamers can be used to create RNAs that self-assemble and create RNA and RNA–protein assemblies in cells. We find that incorporation of five copies of Corn, a dimeric fluorogenic RNA aptamer, into an RNA causes the RNA to form large clusters in cells, reflecting multivalent RNA–RNA interactions enabled by these RNAs. Here, we also describe a second dimeric fluorogenic aptamer, Beetroot, which shows partial sequence similarity to Corn. Both Corn and Beetroot form homodimers with themselves but do not form Corn–Beetroot heterodimers. We thus use Corn and Beetroot to encode distinct RNA–protein assemblies in the same cells. Overall, these studies provide an approach for inducing RNA self-assembly, enable multiplexing of distinct RNA assemblies in cells, and demonstrate that proteins can be recruited to RNA assemblies to genetically encode intracellular RNA–protein assemblies.
Aptamer
Cite
Citations (29)
Cite
Citations (117)
The content, size, and mechanism of synthesis of 3'-terminal poly(A) on the various intracellular species of poliovirus RNA have been examined. All viral RNA species bound to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. At 3 h after infection, the poly(A) on virion RNA, relicative intermediate RNA, polyribosomal RNA, and total cytoplasmic 35S RNA was heterogeneous in size with an average length of 75 nucleotides. By 6 h after infection many of the intracellular RNA's had poly(A) of over 150 nucleotides in length, but the poly(A) in virion RNA did not increase in size suggesting that the amount of poly(A) which can be encapsidated is limited. At all times, the double-stranded poliovirus RNA molecules had poly(A) of 150 to 200 nucleotides. Investigation of the kinetics of poly(A) appearance in the replicative intermediate and in finished 35S molecules indicated that poly(A) is the last portion of the 35S RNA to be synthesized; no nascent poly(A) could be detected in the replicative intermediate. Although this result indicates that poliovirus RNA is synthesized 5' leads to 3' like other RNA's, it also suggests that much of the poly(A) found in the replicative intermediate is an artifact possibly arising from the binding of finished 35S RNA molecules to the replicative intermediate during extraction. The addition of poly(A) to 35S RNA molecules was not sensitive to guanidene.
Nuclease protection assay
Cite
Citations (89)
Cite
Citations (211)
Ultrafiltration (renal)
Riboswitch
Nucleic acid structure
Cite
Citations (12)
Four RNA phages, β, Bl, and I isolated at our laboratory, and MS2 isolated in the United States were studied.1) At 37°, plaque types of Bl and I were larger than those of β and MS2. At 45°, Bl and I gave pinhole-type plaques and β and MS2 medium size plaques which were as large as those at 37°2) MS2 infected cells began to lyse in 45min after infection and β infected cells in 55-60min. The cells infected with Bl or I lysed to a negligible extent.3) The four phages were serologically related and arranged in an order of β, MS2, Bl, I by the neutralization experiment.4) Base composition of phage RNAs were studied. In MS2 RNA and β RNA, molar per cent of guanylic acid was larger than that of uridylic acid, and in Bl RNA and I RNA, molar per cent of uridylic acid was larger than that of guanylic acid.5) Hybridization experiments were conducted between a double stranded RNA of β and a single stranded RNA of other phages. The result suggested that the β RNA was highly homologous with MS2 RNA, and to lesser extents with Bl RNA and I RNA.6) Correlation between the serological character and the RNA structure is discussed.
Bacteriophage MS2
Nuclease protection assay
Cite
Citations (2)