Ribonuclease-resistant RNA Research Articles

Preparation and Evaluation of Ribonuclease-Resistant Viral HIV RNA Standards Based on Armored RNA Technology

Background:The human immunodeficiency virus type 1 (HIV-1) is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome (AIDS). The aim of this study was to construct an RNA-positive control based on armored (AR) RNA technology, using HIV-1 RNA as a model.Methods:The MS2 maturase, a coat protein gene (at positions 1765 to 1787) and HIV-1 pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside (IPTG) at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography.Results:The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 101 to 105 was prepared. Real-time PCR assays had a range of detection between 101 and 105. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33.Conclusion:Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory.

Cotranscriptional Production of Chemically Modified RNA Nanoparticles.

RNA nanoparticles consisting of multiple RNA strands of different sequences forming various three-dimensional structures emerge as promising carriers of siRNAs, RNA aptamers, and ribozymes. In vitro transcription of a mixture of dsDNA templates encoding all the subunits of the RNA nanoparticle may result in cotranscriptional self-assembly of the nanoparticle. Based on our experience with production of RNA nanorings, RNA nanocubes, and RNA three-way junctions, we propose a strategy for optimization of the cotranscriptional production of chemically modified ribonuclease-resistant RNA nanoparticles.

Self-Assembling RNA Nanorings Based on RNAI/II Inverse Kissing Complexes

RNA is an attractive biopolymer for nanodesign of self-assembling particles for nanobiotechnology and synthetic biology. Here, we experimentally characterize by biochemical and biophysical methods the formation of thermostable and ribonuclease resistant RNA nanorings previously proposed by computational design. High yields of fully programmable nanorings were produced based on several RNAI/IIi kissing complex variants selected for their ability to promote polygon self-assembly. This self-assembly strategy relying on the particular geometry of bended kissing complexes has potential for developing short interfering RNA delivery agents.

Ribonuclease-resistant RNA controls and standards

Ribonuclease-resistant RNA controls and standards

In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection

Ribonuclease-resistant RNA molecules that bind to infectious human cytomegalovirus (HCMV) were isolated in vitro from a pool of randomized sequences after 16 cycles of selection and amplification. The two ligands (L13 and L19) characterized exhibited high HCMV-binding affinity in vitro and effectively inhibited viral infection in tissue culture. Their antiviral activity was also specific as they only reacted with two different strains of HCMV but not with the related herpes simplex virus 1 and human cells. These two ligands appeared to function as antivirals by blocking viral entry. Ultraviolet (UV) crosslinking studies suggested that L13 and L19 bind to HCMV essential glycoproteins B and H, respectively. Thus, RNA ligands that bind to different surface antigens of HCMV can be simultaneously isolated by the selection procedure. Our study demonstrates the feasibility of using these RNA ligands as a research tool to identify viral proteins required for infectivity and as an antiviral agent to block viral infection.

Ribonuclease-resistant RNA Controls (Armored RNA) for Reverse Transcription-PCR, Branched DNA, and Genotyping Assays for Hepatitis C Virus

Comparison and evaluation of molecular diagnostic assays for the detection and quantification of hepatitis C virus (HCV) RNA have been limited by the lack of RNA controls and calibrators. Armored RNA technology is a means for producing RNA that is completely protected from plasma ribonucleases. This method produces recombinant pseudoviral particles that are noninfectious and contain predefined RNA sequences. A consensus 412-base sequence from the 5'NCR/Core region of HCV subtype 2b was derived from 34 individually sequenced HCV genotype 2b variants. A DNA fragment encoding the consensus HCV-2b sequence was synthesized de novo, cloned, and expressed as an Armored RNA control. The resulting HCV-2b Armored RNA (AR-HCV-2b) contained the complete HCV-2b consensus RNA sequence encapsidated within a protective protein coat. AR-HCV-2b was fully recoverable from human plasma incubated at 4 degrees C for >300 days. The particles were tested in three clinical assay formats: Amplicor HCV Monitor 1.0, Quantiplex HCV RNA 2.0, and INNO-LiPA HCV II. When added into seronegative, nonviremic plasma, AR-HCV-2b showed reproducible signals and linear dilutions in both the Amplicor and Quantiplex assays. AR-HCV-2b was correctly identified as subtype 2b in the INNO-LiPA line probe assay. The HCV-2b Armored RNA control is a versatile, durable, ribonuclease-resistant viral RNA control that is compatible in three different clinical assay formats.

Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards.

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. "Armored RNA" is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at -20 degreesC or for 60 days at 4 degreesC. Additionally, Armored RNA preparations are homogeneous and noninfectious.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

The topography of the 5' end of 16-S RNA in the presence and absence of ribosomal proteins S4 and S20.

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-s RNA in the absence of ribosomal protein. These data yield important information on the topography and organisation of the 5' third of the 16-s RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA.

Polyadenylate-protein complexes in resting and growing 3T3 cells

The properties of the ribonuclease resistant cytoplasmic ribonucleoprotein particles were studied in contact-inhibited and serum induced proliferating 3T3 cells. The RNP particles were fractionated by oligo (dT)-cellulose chromatography and banded in CsSO 4 gradients. The main RNP fraction, eluted with 25% formamide, contained the major ribonuclease resistant RNA sequences in both resting and growing cells. The protein component of this fraction had a molecular weight of about 72,000 in contact-inhibited cells and 81,000 in serum induced cells.

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. 97 ± 2% of the total membrane phosphorus is accounted for as phospholipid phosphorus. Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences. Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight. Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.

Rhinovirus type 14 RNA polymerase complexes

Rhinovirus type 14 RNA-dependent RNA polymerase complexes were isolated from microsomal and soluble fraction of infected KB cells. Maximum activities were measured at at 6 and 7 hours post inoculation (p.i.) for microsomal and soluble polymerases, respectively. Both polymerase activities are considerably reduced by 8 to 9 hours, p.i., and interval in which the in vivo rate of synthesis of viral RNA is maximal. In vitro RNA products of RNA polymerases in both fractions consist of ribonuclease-sensitive and ribonuclease-resistant RNA of heterogeneous sizes. Detergent treatment of the microsomal RNA polymerase(s) did not affect the total amount of RNA synthesized, the proportion of ribonuclease-sensitive RNA synthesized or the size of the RNA products. The data suggest that RV14RNA polymerase complexes are intially associated with membranes but are then irreversibly released into the soluble phase of the cytoplasm; possible explanations for this phenomena are discussed.

Identification of self-complementary virus-specific ribonucleic acid in chick kidney cells infected with chicken embryo lethal orphan virus.

RNA was extracted from primary chicken embryo kidney (CEK) cells infected with chicken embryo lethal orphan (CELO) virus and exposed to a pulse of (5-3H)-uridine late in infection. When this RNA was self-annealed, 4.5% became resistant to pancreatic ribonuclease digestion. The ribonuclease-resistant RNA was isolated by chromatography on Sephadex G-100, and the RNA was found to have the characteristics of a double-stranded molecule of sedimentation coefficient 8S. Half of the column-isolated RNA hybridized to CELO DNA with equal amounts of virus RNA binding to the heavy or light stands of the CELO DNA, indicating the presence of complementary RNA species late in the infectious cycle of CELO.

Denatured helical sites and ribonuclease resistant RNA associated with DNA from the dormant seeds of a higher plant

Denatured helical regions in the DNA of dormant cotton seeds were detected by means of ribonuclease interaction, methylated albumin kieselguhr chromatography and sedimentation analysis. Ribonuclease resistant RNA was also found associated with the dormant seed DNA. The implications of these two findings were discussed with regard to possible binding sites for RNA that stabilizes folded DNA.

Effects of sodium chloride and pancreatic ribonuclease on the rat-brain nuclear particles: the fate of the protein moiety.

Previous results on RNA‐labelled nuclear particles were compatible with a folded ribonucleo‐protein strand structure. In the present experiments, particles were labelled with tritiated amino acids or with tritiated amino acids and [32P]orthophosphate in vivo; they were prepared by sonication of nuclei.1. Proteins were released progressively from the particles (ϱ= 1.39 g/ml) as shown by the accumulation of material of ϱ= 1.31–1.35 g/ml after treatment with 0.25 M and 0.4 M NaCl. Concomitantly, high‐density ribonucleoproteins were formed.2. Proteins released from the particles at various NaCl concentrations (up to 2 M) sedimented between 0 and 15 S. 30‐S aggregates corresponding to “informofers” were never detected. Some proteins remained bound to the RNA even after treatment with 2 M NaCl.3. Large protein aggregates contaminated the smallest particles (up to 50–60 S) as shown by the sedimentation properties of the products of extensive ribonuclease digestion and the density of protein‐labelled fractions.4. About half of the protein released from the particles by pancreatic ribonuclease treatment had a low sedimentation coefficient (0–15 S) after formaldehyde fixation. The other half had higher polydisperse sedimentation coefficient, and possibly resulted from artificial cross‐linking. A 30‐S protein peak was not detected.5. The material released from the particles by ribonuclease banded at ϱ= 1.35 g/ml, a higher density than that of free proteins (ϱ= 1.31 g/ml). Treatment with 1 M NaCl after ribonuclease digestion released free proteins, some of which were phosphoproteins (ϱ= 1.31 g/ml) and ribonucleoproteins of higher density. It is suggested that these complexes contain ribonuclease‐resistant RNA.These results confirm and complete our previous ones. They are not compatible with the informofer model of the monoparticle since 30‐S protein aggregates could not be isolated after salt or ribonuclease treatment. They are consistent with the folded ribonucleoprotein strand model which predicts the sequential release of proteins.

Human leukemic cells: Properties of an RNA synthesized in the presence of actinomycin D

RNA biosynthesis in the presence of high concentrations of actinomycin D has been studied, utilizing human cells derived from normal healthy donors, patients with infectious mononucleosis and acute lymphatic leukemia. Actinomycin D-resistant RNA synthesis ranged from 18–38, with a higher percentage in human leukemic (CCRF-CEM; CCRF-HSB) cells, as compared with normal (CCRF-EFB; CCRF-TOH) human lymphocytic cells. The RNA synthesized in the presence of actinomycin D was resistant to ribonuclease (A and T 1); and greater resistance was observed in human leukemic cells as compared to the other lymphocytic cells studied herein. Sucrose density gradient analyses and exclusion column chromatography indicated that this particular ribonuclease-resistant RNA synthesized in the presence of actinomycin D exhibited the sedimentation characteristics of low molecular weight RNA (4S). Polyacrylamide gel electrophoresis analyses indicated that this particular RNA is similar to 4 S RNA, which is synthesized in the absence of actinomycin D. Biochemical and biophysical studies indicate that this particular RNA behaves like double-stranded RNA, and its properties are similar to viral RNA. The nature and apparent presence of this double-stranded viral-like RNA in human leukemic lymphocytes, and its possible implication in ‘neoplastic transformation’ are considered.

Detection of ribonuclease-resistant RNA in chloroplasts of corn leaf tissue infected with maize dwarf mosaic virus

Ribonuclease-resistant RNA was detected in fixed sections of maize dwarf mosaic virus infected corn leaves by staining with acridine orange and observation under ultraviolet light. This RNA was found primarily in chloroplasts of bundle sheath parenchyma cells, as early as 24 hr after inoculation. Ribonuclease-resistant RNA was also found in degenerate plastids of a maize dwarf mosaic virus infected albino mutant of corn and in isolated chloroplasts prepared from infected normal corn. Sections pretreated with protease before nuclease and staining treatments showed increased susceptibility of the ribonuclease-resistant RNA to nuclease attack. The significance of ribonuclease-resistant RNA in chloroplasts is discussed in terms of its relevance to viral synthesis.

Rat liver double-stranded RNA from nuclei and whole cells: Isolation, purification and characterization

1. A purified fraction of ribonuclease-resistant RNA isolated from rat liver whole cells and nuclei was chromatographed on cellulose columns, and a double-stranded RNA fraction isolated. 2. The double-stranded structure of the RNA was firmly defined by its behaviour on cellulose, its symmetrical base composition, immunoprecipitin reaction, and its characteristic biphasic heat denaturation curve. 3. A nuclear origin for double-stranded RNA was established. It can be synthesized in the presence of actinomycin D. No significant quantities could be detected in the cytoplasm.

Ultraviolet effects on biological function of RNA phage MS2

Ultraviolet effects on the biological function of MS2 phage were investigated. The adsorption of MS2 phage to the host cells is not affected by ultraviolet irradiation of the phage, but the penetration of phage RNA is reduced. Hydrated uridine was detected in the RNA molecules of ultraviolet-irradiated MS2 phage. The sedimentation patterns of the parental phage RNA molecules extracted from Escherichia coli at 12 min after infection with ultraviolet-irradiated phage are heterogeneous in size. However, ribonuclease-resistant RNA duplex (RF) detectable in cells infected with non-irradiated MS2 phage is not found in these cells. The phage RNA in the cells infected by ultraviolet-irradiated MS2 phage is fragmented into small size molecules even when the infected cells were incubated with chloramphenicol; no degradation of parental phage RNA is observed in cells infected with non-irradiated MS2 phage under the same conditions. Ultraviolet-irradiated MS2 phage also might be able to induce an RNA replicating enzyme (RNA replicase) in the host cells, although the enzymic activity seems to be lower than that induced in the cells infected with non-irradiated MS2 phage.

Japanese encephalitis virus replication: a procedure for the selective isolation and characterization of viral RNA species.

The viral RNA species synthesized in a porcine kidney cell line, PS(Y-15), by Japanese encephalitis virus (JEV) are described. Virus titers on these cells ranged between 106 to 107 p.f.u./ml at the end of 2 to 3 days incubation at 35° C. Actinomycin D (AD) could not be used to unmask JEV RNA synthesis since it inhibited virus replication at concentrations necessary to substantially reduce host cell RNA synthesis. Treatment of cells with 1 μg AD/ml and removal prior to infection permitted good JEV replication, and at the same time strongly suppressed synthesis of 28 S and 18 S cellular ribosomal RNA. The problem of separating viral RNA from non-ribosomal RNA that was still being synthesized by AD pretreated cells was resolved by the isolation of the cytoplasmic membrane fraction of infected cells. RNA extracted from the membranes of infected AD pretreated cells and analyzed for sedimentation characteristics on sucrose gradients has four RNA species not found in uninfected cells.They are: (1) 45 S single stranded RNA believed to be the infectious RNA found in the virion; (2) a 27 S RNA single stranded RNA; (3) a 20 S ribonuclease resistant RNA believed to be double stranded and (4) an 8 S RNA species. The RNA species found in JEV infected cells, except for the 8 S form, have been found in group A arboviruses. The procedure described utilizing AD pretreatment of host cells and the separation of the cellular cytoplasmic fraction may well have value for the study of the biosynthetic events involved in the replication of other animal viruses that are inhibited by AD.