Addition Of RNase Research Articles

An optimised protocol for the expression and purification of adenovirus core protein VII

Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems. The One Shot BL21 Star (DE3) proved favourable over Rosetta 2 (DE3) pLysS with consistent levels of expression between IPTG-induced and auto-induction. We demonstrated that combining chemical and mechanical cell lysis is possible and highly effective. Other noteworthy benefits were observed in using RNase during sample processing. The addition of RNase has significantly improved the quality and quantity of the purified protein as confirmed by chromatographic and western blot analyses. These findings established a solid groundwork for pVII purification methodologies and carry the significant potential to assist in unveiling the core structure of pVII, its arrangement within the core, DNA condensation intricacies, and potential pathways for nuclear transport.

An amplification-free CRISPR/Cas12a-based fluorescence assay for ultrasensitive detection of nuclease activity

Nuclease, such as RNase H and DNase I, plays key roles in plenty of cellular processes and could be potential therapeutic target for drug development. It is necessary to establish rapid and simple-to-use methods to detect nuclease activity. Herein, we develop a Cas12a-based fluorescence assay without any nucleic acid amplification steps for ultrasensitive detection of RNase H or DNase I activity. By our design, the pre-assembled crRNA/ssDNA duplex triggered the cleavage of fluorescent probes in the presence of Cas12a enzymes. However, the crRNA/ssDNA duplex was selectively digested with the addition of RNase H or DNase I, which leaded to fluorescence intensity changes. Under optimized conditions, the method exhibited good analytical performance, achieving a limit of detection (LOD) as low as 0.0082 U/mL for RNase H and 0.13 U/mL for DNase I, respectively. The method was feasible for analysis of RNase H in human serum and cell lysates, as well as for screening of enzyme inhibitors. Moreover, it can be adopted to image RNase H activity in living cells. Together, this study provides a facile platform for nuclease detection and could be expanded for other biomedical research and clinical diagnostics.

High-speed DNA-based rolling motors powered by RNase H.

DNA-based machines that walk by converting chemical energy into controlled motion could be of use in applications such as next generation sensors, drug delivery platforms, and biological computing. Despite their exquisite programmability, DNA-based walkers are, however, challenging to work with due to their low fidelity and slow rates (~1 nm/min). Here, we report DNA-based machines that roll rather than walk, and consequently have a maximum speed and processivity that is three-orders of magnitude greater than conventional DNA motors. The motors are made from DNA-coated spherical particles that hybridise to a surface modified with complementary RNA; motion is achieved through the addition of RNase H, which selectively hydrolyses hybridised RNA. Spherical motors move in a self-avoiding manner, whereas anisotropic particles, such as dimerised particles or rod-shaped particles travel linearly without a track or external force. Finally, we demonstrate detection of single nucleotide polymorphism by measuring particle displacement using a smartphone camera.

Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection

A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (∼5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50°C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB–RNase H scheme can be applied to a broad range of nucleic acid amplification methods.

Real Time Reverse Transcriptase-PCR to Detect Viable Enterobacteriaceae in Milk

This study was conducted to develop a real time reverse transcriptase-PCR (RT-PCR) method for the detection of viable Enterobacteriaceae in milk using primers based on the genes of ribosomal proteins S11 and S13 and to determine effects of heating and subsequent treatments on the threshold cycle (Ct) of the real time RT-PCR. Total RNA was isolated from 17 strains of bacteria including 11 strains of Enterobacteriaceae suspended in milk using a modified Tri reagent method. SYBR Green Master Mix was added to the RNA and the mixture was subjected to the real time RT-PCR. The Cts of eleven type strains of the Enterobacteriaceae in milk (<TEX>$10^7$</TEX> cells) in the real time RT-PCR ranged from 21.5 to 24.6. However, the Cts of Pseudomonas fluorescens, Acinetobacter calcoaceticus, and three gram-positive bacteria were more than 40. The real time RT-PCR detected as low as <TEX>$10^3$</TEX> cells in agarose gel electrophoresis. The Cts increased from 22.0 to 34.2 when milk samples contaminated with Escherichia coli (<TEX>$10^7$</TEX> cells/mL) were heated at <TEX>$65^{\circ}C$</TEX> for 30 min. In addition, subsequent incubation at <TEX>$37^{\circ}C$</TEX> for 6 and 24 h increased the Cts further up to 36.2 and 37.2, respectively. Addition of RNase A to the bacterial suspension obtained from the heated milk and subsequent incubation at <TEX>$37^{\circ}C$</TEX> for 1 h increased the Cts to more than 40. The results of this study suggests that pretreatment of bacterial cells heated in milk with RNase A before RNA extraction might enhance the ability to differentiate between viable and dead bacteria using real time RT-PCR.

Tumor exosomes: a novel biomarker?

Tumor exosomes: a novel biomarker?

Potential Pitfalls In Quantitative Real-Time PCR for Molecular Monitoring of BCR-ABL In the Management of CML.

AbstractAbstract 1228Due to a lack of standardized methodologies and reporting, results of BCR-ABL transcripts by quantitative real-time PCR (qPCR) are difficult to evaluate and compare between laboratories in molecular monitoring of BCR-ABL in CML. Although progress has been made in harmonization of reporting BCR-ABL quantification, such as the introduction of the International Scale, the standardization of each step in qPCR for BCR-ABL quantification is important. The majority of laboratories use an absolute approach with a standard curve to quantitate the transcript level of BCR-ABL. A number of sources are available for standards, such as RNA, cDNA, and plasmid. Among those, plasmid DNA is the most widely used. Since the standard is the most important factor in the absolute approach, in this study different preparations of plasmid DNA were investigated by qPCR with a TaqMan probe to evaluate their amplification rate. BCR-ABL b3a2 and ABL were individually cloned into pGEM-T Easy vector, and each plasmid DNA was isolated with Qiagen Plasmid Maxi kit. After quantification, DNA was diluted to obtain E6 copy number as templates for qPCR. For data comparison, a common threshhold was set in the middle of the exponential phase and Ct values were obtained. A small Ct value indicates a high amplification rate. Multiple preparations of purified completely linearized plasmid DNA for both b3a2 and ABL have the Ct values of 17.90 ± 0.19 and 17.94 ± 0.44 respectively. These values were very similar or equal to the Cts of purified b3a2 and ABL DNA fragments derived from RT-PCR. However, the non-linearized circular plasmid DNA only had 20.36 ± 0.08 for b3a2 and 20.31 ± 0.13 for ABL when the same number of templates was analyzed in the same qPCR indicating a 5.5 fold deficiency in amplification rate. Although the amplification efficiency obtained from both linearized and circular plasmids were very similar in the range of 3.3±0.3, a deficiency in the amplification rate from the circular plasmid indicates that the complicated super coiled structure in the circular plasmid DNA may interfere with the amplification. After plasmid DNA was completely linearized with three individual restriction enzymes, aliquots of known amounts of plasmid DNA were adjusted to E6 copy number and analyzed without further purification. Depending on the restriction enzyme the amplification rate of non-purified plasmid varied from 69 to almost 100% compared with purified linearized plasmid DNA. To mimic the purification procedure, Rnase A was added to the restriction digestion mixture to remove RNA contamination. Without purification, the addition of RNase A significantly inhibits the amplification rate. Furthermore, standards from Ipsogen were compared in the study. From multiple concentration points in comparison of the standards, Ipsogen standards had a Ct value of 1.43 more than the purified completely linearized plasmid DNA with 2.7 fold deficiency in amplification rate. Since the Ct value of the sample is the same, the standards with low amplification rate would over-estimate the copy number of the sample. In theory, the copy ratio of the fusion gene (b3a2) to the control gene (ABL) should be the same, independent on the amplification rate of the standards. However, in practical experiments with a high amplification rate of the standards, the copy ratio is more accurate and reproducible when the samples have a low copy number of BCR-ABL. The combination of incomplete linearization of plasmid DNA in restriction enzyme digestion, non-purification of the linearized plasmid and contamination of interfering substances may together decrease the amplification rate. The preparation of purified completely linearized plasmid provides a standard way to obtain standards for qPCR in molecular monitoring of the BCR-ABL transcript level in the management of CML patients as well as for qPCR in other usages. Disclosures:No relevant conflicts of interest to declare.

G4-forming Sequences in the Non-transcribed DNA Strand Pose Blocks to T7 RNA Polymerase and Mammalian RNA Polymerase II

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

Novel Vybrant® DyeCycle ™ Stains Provide Cell Cycle Analysis in Live Cells Using Flow Cytometry with Violet, Blue, and Green Excitation.

Flow cytometric approaches can resolve cell position in the cell cycle based on DNA content, and these analyses are widely used in the study of cell growth, cell cycle regulation, and oncology. These applications require dyes that bind to DNA in a stoichiometric manner and which, with the exception of some UV-excited dyes like Hoechst 33342, require fixation, permeabilization and RNAse treatment for reproducible results. The Vybrant® DyeCycle™ stains are DNA-selective, cell membrane-permeant dyes that show greatly-enhanced fluorescence when bound to DNA. DyeCycle violet stain can be excited by a 405 nm laser, DyeCycle green stain by a 488 nm laser, and DyeCycle orange stain by either 488 nm or 532 nm lasers. (Figure 1) No fixation, permeabilization or addition of RNAse is required for stoichiometric binding of the dye to DNA. These dyes show similar performance on live Jurkat cells to Hoechst 33342 and DRAQ5: G0/G1 peak CV generally less than 6% and G2/G1 ratio greater than 1.8. The DyeCycle stains have been tested on a variety of cells: Jurkat, CHO, NIH 3T3, HL60, HEK cells, and peripheral blood leukocytes. Staining was optimized by cell type using time, temperature, cell concentration and dye concentration. The dyes can be used on cells in the presence of media components, including serum and divalent cations. Cells with damaged membranes exhibit different DyeCycle stain uptake patterns from live cells and can be excluded from analysis with spectrally-resolved viability dyes, such as SYTOX® blue and SYTOX red dyes. The DyeCycle stains have been used with antibody staining against surface antigens to evaluate the cycle profile of subpopulations. Generally, antibody conjugates with red-emitting fluorophores, such as allophycocyanin and phycoerythrin tandem fluorophores, were used because the required concentrations of the DyeCycle stains produced significant emission across fluorescein and phycoerythrin detection channels. The DyeCycle stains have also been combined with viability and apoptotic markers in testing of cells induced to apoptosis with camptothecin. DyeCycle orange stain, in particular, was found to define a sub-G0 population in late apoptotic cells. Finally, the DyeCycle stains cause some retardation of cell division, but do not demonstrate the toxicity observed with DRAQ5. The adherent cell lines, HEK and NIH 3T3, were stained with DyeCycle violet stain and DyeCycle orange stain, respectively before being were sorted under sterile conditions based on G0/G1 and G2/M population fluorescence. Sorted populations demonstrated the appropriate fluorescence when verified by reanalysis, and all sorted populations attached normally and expanded over 1 to 3 days post-sort. DyeCycle stains allow resolution of cell cycle information in viable cells against the dynamic background of cell activity using common lasers, as well as the ability to sort cells based on position in the cell cycle. [Display omitted]

Essential Role of KIBRA in Co-activator Function of Dynein Light Chain 1 in Mammalian Cells

Recently dynein light chain 1 (DLC1), a cytoskeleton signaling component, has been shown to interact with and transactivate estrogen receptor-alpha (ER), leading to increased expression of ER target genes and growth stimulation of breast cancer cells. However, the molecular mechanism by which DLC1 regulates the ER pathway remains poorly understood. To gain insights into the putative mechanism, here we set out to identify novel DLC1-interacting proteins. We identified KIBRA, a WW domain- and a glutamic acid stretch-containing protein, as a DLC1-binding protein and showed that it interacts with DLC1 both in vitro and in vivo. We found that KIBRA-DLC1 complex is recruited to ER-responsive promoters. We also found that KIBRA-DLC1 interaction is mandatory for the recruitment and transactivation functions of ER or DLC1 to the target chromatin. Finally we found that KIBRA interacts with histone H3 via its glutamic acid-rich region and that such interaction might play a mechanistic role in conferring an optimal ER transactivation function as well as the proliferation of ligand-stimulated breast cancer cells. Together these findings indicate that DLC1-KIBRA interaction is essential for ER transactivation in breast cancer cells.

RNA Selectively Interacts with Vesicles Depending on Their Size

RNA and vesicles are two important molecular classes in the origin of life and early evolution, but they are not generally considered as interacting partners. The present paper reports about the interaction between tRNA (Esherichia coli) and vesicles made of the zwitterionic surfactant POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), partially positively charged with small molar fractions (max 10%) of the single-chained CTAB (cetyltrimethylammonium bromide). CTAB is capable to insert efficiently in POPC vesicles (as determined by zeta-potential measurements), and the binding of tRNA to such charged vesicles operates a strong selection being critically dependent upon the vesicle size. The binding of tRNA to the vesicles is size-selective as it induces a strongly pronounced process of aggregation of large vesicles (ca. 160-nm diameter) but not of small ones (ca. 80-nm diameter) that are stable against vesicle aggregation (as followed by dynamic light-scattering and optical density measurements). The aggregation of the large vesicles is fully reversible upon the addition of RNase A. The selective behavior of tRNA with respect to differently sized vesicles is observable in separated samples as well as in a mixture of both populations. In the latter case, the fraction of large vesicles readily aggregates in the presence of the small ones that remain unaltered in the mixture. This kind of discrimination capability of RNA might have been of importance in the early phases of the formation of the protocells.

Transcription arrest caused by long nascent RNA chains

The transcription process is highly processive. However, specific sequence elements encoded in the nascent RNA may signal transcription pausing and/or termination. We find that under certain conditions nascent RNA chains can have a strong and apparently sequence-independent inhibitory effect on transcription. Using phage T3 RNA polymerase (T3 RNAP) and covalently closed circular (cccDNA) DNA templates that did not contain any strong termination signal, transcription was severely inhibited after a short period of time. Less than ∼10% residual transcriptional activity remained after 10 min of incubation. The addition of RNase A almost fully restored transcription in a dose dependent manner. Throughout RNase A rescue, an elongation rate of ∼170 nt/s was maintained and this velocity was independent of RNA transcript length, at least up to 6 kb. Instead, RNase A rescue increased the number of active elongation complexes. Thus transcription behaved as an all-or-none process. The mechanism of transcription inhibition was explored using electron microscopy and further biochemical experiments. The data suggest that multiple mechanisms may contribute to the observed effects. Part of the inhibition can be ascribed to the formation of R-loops between the nascent RNA and the DNA template, which provides “roadblocks” to trailing T3 RNAPs. Based on available literature we discuss possible in vivo implications of the results.

The use of reverse transcriptase for efficient first- and second-strand cDNA synthesis from single- and double-stranded RNA templates

Molecular characterization of eight distinct, difficult-to-clone RNA plant viruses was accomplished after the development of a reverse transcriptase-based first- and second-strand cDNA synthesis method. Double-stranded (ds) RNA templates isolated from strawberry and blackberry and several herbaceous hosts (mint, pea and tobacco) were cloned using this method. Templates, combined with random primers, were denatured with methyl mercuric hydroxide. Reverse transcriptase was added followed by the addition of RNase H. The resulting dsDNA was then digested with restriction endonucleases to produce shorter fragments that could be cloned efficiently into a T-tailed vector after adding an A-overhang using Taq polymerase. This procedure resulted in a high number of cloned fragments and allowed insert sizes up to three kilobase-pairs. Unlike traditional cDNA construction methods, there is no need for additional enzymes/steps for second-strand synthesis, PCR amplification or prior sequence information. Synthesis and cloning of cDNA derived from dsRNA templates is much more efficient than with previously described methods. This procedure also worked well for cloning gel-purified dsRNA and with single-stranded RNA templates.

Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we observed a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 microM tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 microM dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants.

A sensitive method for the detection of enterotoxigenic Escherichia coli by the polymerase chain reaction using multiple primer pairs

In this study, a polymerase chain reaction (PCR) method has been developed for the detection of enterotoxigenic Escherichia coli (ETEC). Three different sets of oligonucleotide primers synthesized were used to amplify the enterotoxin genes of heat-labile (LTh) and heat-stable (STIa and STIb) enterotoxins of ETEC. These primers amplified a 627, 240, or 169 base pair (bp) DNA fragment from LTh, STIa and STIb gene, respectively, of the reference ETEC strains. The addition of RNase A (10 micrograms/ml) to the PCR reaction solution diminished nonspecific amplification of DNA fragments other than the enterotoxin genes. Five types of ETEC strains corresponding to the LTh, STIa, STIb, LTh-STIa, or LTh-STIb genotypes were distinguished by a single procedure of PCR using the mixture of the three sets of primers. PCR, hybridization, and conventional methods were subjected to one hundred stool specimens from diarrheal patients. It was found that PCR was the most sensitive method among them. These results suggested that PCR with triple primer pairs would be useful for the laboratory diagnosis of ETEC in the stool specimens.

Preparation and characterization of monoclonal antibodies specific for ribonuclease inhibitor from rat liver.

Monoclonal antibodies specific for ribonuclease inhibitor (RNasin) were obtained by the method of production of hybridomas from spleen cells and myeloma cells. The specificity of monoclonal antibodies obtained by cloning was investigated by Western blot analysis. The addition of ribonuclease (RNase) A to RNasin resulted in the inhibition of antibody binding to the antigen. However, the addition of RNase A to the antigen-antibody complex did not cause dissociation of the complex.

The synthesis of oligodeoxyribonucleotides using RNA ligase

T4 RNA ligase catalyzes the addition of a single deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of oligodeoxyribonucleotides (Hinton et al. (1978) Biochemistry 17, 5091). We have determined improved conditions for this reaction which give yields equal to or greater than 85% when any of five common deoxyribonucleoside bisphosphate (pdAp, pdCp, pdGp, pdTp, or pdUp) are added to dA(PDA)4. A low ATP concentration, which is constantly maintained by a regeneration system composed of phosphocreatine, creatine kinase, and myokinase, contributes to the attainment of high yields. The addition of RNase A and spermine also enhances the rates and yields of the reactions. These conditions facilitate the use of RNA ligase as a reagent for the stepwise synthesis of DNA of defined sequence.

Chromatin- and Nuclei-Directed Ribonucleic Acid Synthesis in Sugar Beet Root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg(2+) or Mn(2+)) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.Washing tissue in solutions of gibberellic acid and auxin enhances template availability of the isolated chromatin. Experiments with isolated nuclei indicate an effect of these hormones on RNA synthesis.

The effect of ribonuclease on the binding of calcium by mitochondria

Mitochondria, isolated from potato tuber, pretreated with ribonuclease (RNase) showed an increase in calcium binding at low enzyme concentration. The same dosage-response pattern was obtained whether the enzyme treatment was conducted for 10, 30 or 120 min. However, when the enzyme treatment was carried out at 0° instead of at 30°, no increase in Ca-binding was obtained, suggesting that no interaction occurred between the enzyme and the divalent cation. Oxygen consumption under the same conditions was not affected. Microsomes treated with RNase did not show a change in their Ca-binding capacity, although untreated microsomes showed about the same increase in Ca-binding with increase in temperature as did the mitochondria. When mitochondria prelabeled with (45)Ca, after extracting their inorganic calcium phosphate, were treated with RNase a liberation of Ca, ribonucleotide, and phosphate was observed. It is suggested that Ca ions form a bridge like bond between ribonucleic acid and either phospholipids or phospholipoproteins, because RNase liberated more phosphate than nucleotides and the extra phosphate cannot be inorganic calcium phosphate, since the calcium phosphate was extracted before addition of RNAse.