RNA Lysates Research Articles

High-Efficiency 3D DNA Walker Immobilized by a DNA Tetrahedral Nanostructure for Fast and Ultrasensitive Electrochemical Detection of MiRNA.

Herein, by directly limiting the reaction space, an ingenious three-dimensional (3D) DNA walker (IDW) with high walking efficiency is developed for rapid and sensitive detection of miRNA. Compared with the traditional DNA walker, the IDW immobilized by the DNA tetrahedral nanostructure (DTN) brings stronger kinetic and thermodynamic favorability resulting from its improved local concentration and space confinement effect, accompanied by a quite faster reaction speed and much better walking efficiency. Once traces of target miRNA-21 react with the prelocked IDW, the IDW could be largely activated and walk on the interface of the electrode to trigger the cleavage of H2 with the assistance of Mg2+, resulting in the release of amounts of methylene blue (MB) labeled on H2 from the electrode surface and the obvious decrease of the electrode signal. Impressively, the IDW reveals a conversion efficiency as high as 9.33 × 108 in 30 min with a much fast reaction speed, which is at least five times beyond that of typical DNA walkers. Therefore, the IDW could address the inherent challenges of the traditional DNA walker easily: slow walking speed and low efficiency. Notably, the IDW as a DNA nanomachine was utilized to construct a sensitive sensing platform for rapid miRNA-21 detection with a limit of detection (LOD) of 19.8 aM and realize the highly sensitive assay of biomarker miRNA-21 in the total RNA lysates of cancer cell. The strategy thus helps in the design of a versatile nucleic acid conversion and signal amplification approach for practical applications in the areas of biosensing assay, DNA nanotechnology, and clinical diagnosis.

Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs.

ABSTRACTAlphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons. Differentiated AP-7 rat olfactory neuronal cells, an in vitro model for mature neurons, were infected with SINV and treated with anti-E2 antibody. Whole-cell RNA lysates were collected for sequencing of poly(A)-selected RNA 24, 48, and 72 h after infection. Three primary species of viral RNA were produced: genomic, subgenomic, and defective viral genomes (DVGs) encoding the RNA capping protein nsP1. Antibody treatment resulted in overall lower production of SINV RNA, decreased synthesis of subgenomic RNA relative to genomic RNA, and suppressed production of the nsP1 DVG. The nsP1 DVG was packaged into virus particles and could be translated. Because antibody-treated cells released a higher proportion of virions with noncapped genomes and transient transfection to express the nsP1 DVG improved viral RNA capping in antibody-treated cells, we postulate that one mechanism by which antibody inhibits SINV replication in neurons is to suppress DVG synthesis and thus decrease production of infectious virions containing capped genomes.

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model.

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.

The Noncoding RNA nc886 Regulates PKR Signaling and Cytokine Production in Human Cells.

Protein kinase RNA-activated (PKR) is a cytoplasmic receptor for dsRNA, and as such is involved in detection of viral infection. On binding dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates its substrate, eukaryotic translation initiation factor 2 subunit α (eIF2α), causing inhibition of mRNA translation and shutdown of viral protein production. However, active PKR has also been found to be involved in the NF-κB signaling pathway by inducing phosphorylation of IκBα. PKR is regulated by the noncoding RNA nc886, which has altered expression in cancer. We have found that expression of nc886 is highly upregulated during activation of human CD4+ T cells. As has been described in other cell types, nc886 bound to PKR in human T cell lysates, preventing PKR phosphorylation by polyinosinic:polycytidylic acid or HIV trans-activation response element RNA in lysates of T cell lines or primary human CD4+ T cells. Using clonal human T cell lines, we found that nc886 expression was strictly required for IFN-γ and IL-2 expression and secretion after T cell activation but did not affect proliferation or activation-induced cell death. In stimulated human PBMCs, nc886 expression strongly correlated with IFN-γ expression. Although nc886 inhibited PKR activation by dsRNA, it was required for PKR phosphorylation during T cell stimulation, with subsequent NF-κB signaling and CREB phosphorylation. nc886 also regulated PKR phosphorylation during human monocyte-derived macrophage activation. We have therefore identified nc886 as a noncoding RNA marker of T cell activation and regulator of PKR-dependent signaling.

Ultrasensitive SPR detection of miRNA-93 using antibody-enhanced and enzymatic signal amplification.

MiRNAs are endogenous noncoding RNAmolecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present an SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10pmol/L was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1fmol/L.

ATP-binding cassette transporters in immortalised human brain microvascular endothelial cells in normal and hypoxic conditions

BackgroundRapid reperfusion following ischemia is the most effective therapy in stroke therapy. However, the success may be compromised by ischemia & reperfusion (I/R) injury and at the human blood–brain barrier (BBB), therefore the effects on transendothelial transport are of special interest. Current studies suggest the ATP-binding cassette (ABC) transporters to be regulated upon ischemic stroke in a way that impedes the effects of drug therapy. The immortalised human brain microvascular endothelial cell line hCMEC/D3 provides most of the unique properties of the BBB with respect to transport and might be a reliable in vitro model to study transendothelial transport after I/R.MethodsWe exposed hCMEC/D3 cells to 24 hours of hypoxia alone and to hypoxia followed by 60 min of reoxygenisation as an in vitro model for I/R. Western blot showed mild upregulation of hypoxia inducible factor (HIF-1α) after hypoxia alone and RNA lysates were analysed with a well-established real-time RT-PCR-based TaqMan low-density array detecting 47 of 48 known human ABC transporters.ResultsNo significant increases of ABC mRNA expression levels were detected neither in hypoxic nor in I/R samples. However, slight decrease of ABCC1 in hypoxic and I/R samples and of ABCA10 and ABCD3 in I/R samples was observed.ConclusionOur data suggests that hCMEC/D3 cell line and – at the moment – in vitro models in general are a poor basis for stroke research but may be enhanced by co-culturing more cells of the neurovascular unit inducing an overall ischemic response at the BBB.

Effect of Topical Microbicides on Infectious Human Immunodeficiency Virus Type 1 Binding to Epithelial Cells

Topical microbicides (cellulose acetate 1,2 benzene dicarboxylate [CAP], PRO 2000, SPL7013, and UC781) are being investigated to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1). These products were shown to prevent the transfer of infectious HIV-1 from urogenital and colorectal epithelial cell lines to peripheral blood mononuclear cells. However, it was unclear if the topical microbicides rendered the virus noninfectious and/or reduced the binding to the epithelial cells. To test this, epithelial cells were cultured with HIV-1 in the presence or absence of topical microbicides or their placebos. The cells were washed, RNA lysates were made, and real-time PCR was performed for HIV-1. PRO 2000 and SPL7013 significantly (P < 0.0001) reduced the amount of bound HIV-1 to the colorectal epithelial cell line across clades A, B, C, and CRF01-AE. While none of the products reduced the binding of HIV-1 clades A and C to the urogenital cell line, CAP, PRO 2000, and SPL7013 significantly (P </= 0.002) reduced the binding of clades B and CRF01-AE. In general, PRO 2000 and SPL7013 placebos significantly (P < 0.0001) reduced the amount of bound HIV-1 but were less than the active products. UC781, its placebo, and hydroxyethyl cellulose (placebo for CAP) minimally affected the amount of bound HIV-1. These results suggest that rendering HIV-1 noninfectious may not correlate to the amount of HIV-1 bound to epithelial cells and possible shedding into mucosal secretions. Therefore, functional virological assays in addition to measuring viral RNA should be included when clinically evaluating topical microbicide use by infected persons.

DNA binding domain-independent pathways are involved in EWS/FLI1-mediated oncogenesis.

Specific chromosomal translocations involving the ews gene and one of five members of the ets family of transcription factors create ews/ets fusion genes that are found in approximately 85% of Ewing's family of tumors. ews/ets fusion genes consistently maintain an intact and functional ets DNA binding domain (DBD) in all of these cases. We demonstrate here, however, that EWS/FLI1, the most prevalent EWS/ETS fusion, activates oncogenic pathways independent of its DBD. In in vivo tumor assays, EWS/FLI1 molecules with either point mutations or a large deletion in the ets DBD retain the ability to accelerate tumors in NIH 3T3 cells, whereas they lose the ability to bind DNA in vitro. Additionally, whereas inhibition of DBD functions of EWS/FLI1 with a dominant negative form of FLI1 is sufficient to inhibit anchorage-independent growth in NIH 3T3 cells, it is ineffective in inhibiting tumor growth in SCID mice. Usage of this dominant negative construct in a Ewing's tumor cell line, however, does reduce the rate of tumor formation, supporting the need for a functional DBD in this context. Together, these results suggest that EWS/FLI1 induces both DBD-dependent and DBD-independent oncogenic pathways.

Endogenous inhibitors of the dsRNA-dependent eIF-2α protein kinase PKR in normal and ras-transformed cells

The serine/threonine kinase PKR is activated by autophosphorylation in response to nanomolar concentrations of double-stranded RNA and other polyanions. We have previously shown that expression of an oncogenic ras gene induces an endogenous protein inhibitor of PKR activation in murine fibroblasts, as measured by a greatly reduced ability of PKR to autophosphorylate in response to double-stranded RNA in lysates from these ras-expressing cells. Immunoprecipitation of PKR away from the ras-transformed cell lysate restored the ability of PKR to become autophosphorylated. However, the autophosphorylation was no longer dsRNA-dependent. In the present work, PKR immobilized either by immunoprecipitation or by affinity precipitation on Agpoly(I) poly(C) was found to autophosphorylate in a dsRNA-independent manner when incubated in the presence of a detergent lysis buffer and ATP. When lysis buffer was replaced by cytoplasmic extract from normal or ras-transformed cells, autophosphorylation of the immobilized PKR was inhibited in the presence or absence of dsRNA, even though it could be shown that PKR remained bound and intact in the precipitate, and able to autophosphorylate if rewashed with lysis buffer. These findings suggest that PKR activation is regulated by an endogenous inhibitor in murine fibroblasts as well as by dsRNA or other polyanions.

Degradation of Nucleic Acids in Cell Lysates by S1 Nuclease in the Presence of 9 M Urea and Sodium Dodecylsulfate

Single-strand-specific nuclease S1 from Aspergillus oryzae is shown to degrade DNA and RNA in lysates of HeLa cells in the presence of 9 M urea and sodium dodecylsulfate. Free dodecylsulfate inhibits S1 nuclease. However, if the detergent is complexed with proteins prior to the addition of the enzyme, S1 nuclease can degrade nucleic acids at dodecylsulfate concentrations which would inhibit the enzyme completely if no other proteins were present. In lysates prepared from HeLa cells by treatment with dodecylsulfate and urea, the detergent is complexed by cellular proteins and therefore S1 nuclease can be used to digest DNA and RNA. DNA can be completely degraded but, even after heat-denaturation, only 60% of the cellular RNA is converted into acid-soluble material. Analysis of the acid-insoluble RNA fragments by gel filtration reveals that the majority of the degradation products is approximately of tRNA size.

Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes

Sindbis virus-specific polypeptides were synthesized in lysates of rabbit reticulocytes in response to added 26 S or 49 S RNA. Sindbis 26 S RNA was translated into as many as three polypeptides which co-migrate in acrylamide gels with proteins found in infected cells. Wild type 26 S RNA was translated primarily into two polypeptides, which appear to be the Sindbis nucleocapsid protein (mol. wt 30,000) and the precursor of the two glycoproteins of the virion (mol. wt 100,000). A larger polypeptide (mol. wt 130,000) was synthesized in response to ts2 26 S RNA, a species of RNA which was isolated from cells infected with the ts2 mutant of Sindbis virus. This large polypeptide is apparently the protein which accumulates in cells infected with the mutant virus and which is thought to be a precursor of all three viral structural proteins. These results support the hypothesis that 26 S RNA is the messenger for the three structural proteins of the virion and that the RNA codes for one large polypeptide precursor. The precursor may then be cleaved at a specific site to yield the nucleocapsid protein and a second polypeptide which, in infected cells, is cleaved in a series of steps to yield the two glycoproteins of the virion. Sindbis 49 S RNA was translated into eight or nine polypeptides ranging from 60,000 to 180,000 molecular weights. The viral structural proteins, as such, were not synthesized in response to the added 49 S RNA.

The density of ribosomes bearing messenger RNA in phage-infected and normal bacteria.

ABSTRACT Newly made viral RNA in lysates of Escherichia coli infected with phage R17 was studied as a model messenger RNA. The viral RNA that was associated with groups of 1, 2, 3 and 4 ribosomes banded in caesium chloride at densities of approximately 1·68, 1·65, 1·645 and 1·635 g cm-3 respectively and the single ribosomes of uninfected cells at 1·62. The density of infected ribosomes was restored to 1·62 by treatment with RNase. These values are consistent with the presence of a single molecule of viral RNA in each group. In uninfected bacteria the mean density of the smaller polysomes exceeded that of actino-mycintreated or RNase-treated ribosomes by about 0·005 g cm−3. Similarly, pulse-labelled RNA associated with single ribosomes or pairs banded at a density only marginally higher than that of the bulk of the single ribosomes. If any RNA was attached to the ribosomes it probably amounted to not more than one fifth of the size of the phage RNA for each ribosome pair.

On the function and fate of phage progeny RNA in infected bacteria.

ABSTRACT Spheroplasts of Escherichia coli infected with RNA phage R17 were treated with actinomycin D to inhibit the synthesis of host RNA and protein. Ribosomes occurred predominantly as monomers, dimers and trimers which were active in viral protein synthesis. Partly finished strands of viral RNA in lysates sedimented at a rate characteristic of groups of 6–12 ribosomes and their buoyant density suggested that they were in fact attached to ribosomes. Newly completed strands were associated with single (70-s) ribosomes, forming a 75-S complex, and with ribosome dimers and trimers. Some were probably loosely bound to 30-s ribosomal subunits. Newly made RNA began to accumulate in mature virions after a lag of about 5 min and rose to 16 % of the total labelled viral RNA by the end of the growth cycle, either with continuous labelling or after a 5-min pulse followed by a non-radioactive chase. It is suggested that most, if not all, new viral RNA forms a rather stable association with ribosomes and that a small proportion of this RNA may subsequently pass into progeny virus.