Role of detergents and nuclease inhibitors in the extraction of RNA from eukaryotic cells in complex matrices.

The potential for liquid biopsy samples to be used in place of more invasive tissue biopsies has become increasingly revalent as it has been found that nucleic acids (NAs) present in the blood of cancer patients originate from tumors. Nanomagnetic extraction has proven to be a highly effective means to rapidly prepare NA from clinical samples for molecular diagnostics. In this article, the lysis reaction used to extract RNA from the human epithelial melanoma cells have been optimized using silica coated superparamagnetic nanoparticles (SPM NP). The lysis buffer (LB) is composed of several agents that denature cells, i.e., surfactant and guanidinium isothiocyanate (GITC), and agents that inhibit the degradation of circulated nucleic acids (cfNAs). The surfactant Triton X-100 has been widely used in LB but has been placed on the European Union REACH list. We have compared the qRT-PCR sensitivity resulting from LBs composed of Triton X-100 to several sustainable surfactants, i.e., Tergitol 15-S-7, Tergitol 15-S-9 and Tween-20. Surprisingly, the inclusion of these surfactants in the LB was not found to significantly improve cell lysis, and subsequently the sensitivity of qRT-PCR. The role of the sample matrix was also examined by performing extractions from solutions containing up to 30 mg mL-1 serum albumin. The qRT-PCR sensitivity was found to decrease as the concentration of this protein was increased; however, this was linked to an increased RNase activity and not the concentration of the protein itself. These results lead us to recommend a reformulation of LB for clinical samples, and to conclude that sensitive qRT-PCR RNA analysis can be performed in serum with the timely addition of an RNase inhibitor.

Barnase-Barstar Pair: Contemporary Application in Cancer Research and Nanotechnology.

Barnase is an extracellular ribonuclease secreted by Bacillus amyloliquefaciens that was originally studied as a small stable enzyme with robust folding. The identification of barnase intracellular inhibitor barstar led to the discovery of an incredibly strong protein-protein interaction. Together, barnase and barstar provide a fully genetically encoded toxin-antitoxin pair having an extremely low dissociation constant. Moreover, compared to other dimerization systems, the barnase-barstar module provides the exact one-to-one ratio of the complex components and possesses high stability of each component in a complex and high solubility in aqueous solutions without self-aggregation. The unique properties of barnase and barstar allow the application of this pair for the engineering of different variants of targeted anticancer compounds and cytotoxic supramolecular complexes. Using barnase in suicide gene therapy has also found its niche in anticancer therapy. The application of barnase and barstar in contemporary experimental cancer therapy is reflected in the review.

Reprogramming of Protein-Targeted Small-Molecule Medicines to RNA by Ribonuclease Recruitment.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

Delayed Processing of Secretin-Induced Pancreas Fluid Influences the Quality and Integrity of Proteins and Nucleic Acids.

OBJECTIVES: Endoscopic pancreatic function tests are used to diagnose pancreatic diseases and are a viable source for the discovery of biomarkers to better characterize pancreatic disorders. However, pancreatic fluid (PF) contains active enzymes that degrade biomolecules. Therefore, we tested how preservation methods and time to storage influence the integrity and quality of proteins and nucleic acids. METHODS: We obtained PF from 9 subjects who underwent an endoscopic pancreatic function test. Samples were snap frozen at the time of collection; after 1, 2, and 4 hours on ice; or after storage overnight at 4°C with or without RNase or protease inhibitors (PIs). Electrophoresis and mass spectrometry analysis determined protein abundance and quality, whereas nucleic acid integrity values determined DNA and RNA degradation. RESULTS: Protein degradation increased after 4 hours on ice and DNA degradation after 2 hours on ice. Adding PIs delayed degradation. RNA was significantly degraded under all conditions compared with the snap frozen samples. Isolated RNA from PF-derived exosomes exhibited similar poor quality as RNA isolated from matched PF samples. CONCLUSIONS: Adding PIs immediately after collecting PF and processing the fluid within 4 hours of collection maintains the protein and nucleic acid integrity for use in downstream molecular analyses.

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

Protein-responsive protein release of supramolecular/polymer hydrogel composite integrating enzyme activation systems.

Non-enzymatic proteins including antibodies function as biomarkers and are used as biopharmaceuticals in several diseases. Protein-responsive soft materials capable of the controlled release of drugs and proteins have potential for use in next-generation diagnosis and therapies. Here, we describe a supramolecular/agarose hydrogel composite that can release a protein in response to a non-enzymatic protein. A non-enzymatic protein-responsive system is developed by hybridization of an enzyme-sensitive supramolecular hydrogel with a protein-triggered enzyme activation set. In situ imaging shows that the supramolecular/agarose hydrogel composite consists of orthogonal domains of supramolecular fibers and agarose, which play distinct roles in protein entrapment and mechanical stiffness, respectively. Integrating the enzyme activation set with the composite allows for controlled release of the embedded RNase in response to an antibody. Such composite hydrogels would be promising as a matrix embedded in a body, which can autonomously release biopharmaceuticals by sensing biomarker proteins.

Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor.

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.

Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs.

Ribonucleases (RNases) are a large number of enzymes gathered into different bacterial or eukaryotic superfamilies. Bovine pancreatic RNase A, bovine seminal BS-RNase, human pancreatic RNase 1, angiogenin (RNase 5), and amphibian onconase belong to the pancreatic type superfamily, while binase and barnase are in the bacterial RNase N1/T1 family. In physiological conditions, most RNases secreted in the extracellular space counteract the undesired effects of extracellular RNAs and become protective against infections. Instead, if they enter the cell, RNases can digest intracellular RNAs, becoming cytotoxic and having advantageous effects against malignant cells. Their biological activities have been investigated either in vitro, toward a number of different cancer cell lines, or in some cases in vivo to test their potential therapeutic use. However, immunogenicity or other undesired effects have sometimes been associated with their action. Nevertheless, the use of RNases in therapy remains an appealing strategy against some still incurable tumors, such as mesothelioma, melanoma, or pancreatic cancer. The RNase inhibitor (RI) present inside almost all cells is the most efficacious sentry to counteract the ribonucleolytic action against intracellular RNAs because it forms a tight, irreversible and enzymatically inactive complex with many monomeric RNases. Therefore, dimerization or multimerization could represent a useful strategy for RNases to exert a remarkable cytotoxic activity by evading the interaction with RI by steric hindrance. Indeed, the majority of the mentioned RNases can hetero-dimerize with antibody derivatives, or even homo-dimerize or multimerize, spontaneously or artificially. This can occur through weak interactions or upon introducing covalent bonds. Immuno-RNases, in particular, are fusion proteins representing promising drugs by combining high target specificity with easy delivery in tumors. The results concerning the biological features of many RNases reported in the literature are described and discussed in this review. Furthermore, the activities displayed by some RNases forming oligomeric complexes, the mechanisms driving toward these supramolecular structures, and the biological rebounds connected are analyzed. These aspects are offered with the perspective to suggest possible efficacious therapeutic applications for RNases oligomeric derivatives that could contemporarily lack, or strongly reduce, immunogenicity and other undesired side-effects.

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus.

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.

The role of monocyte chemotactic protein-induced protein 1 (MCPIP1) in angiotensin II-induced macrophage apoptosis and vulnerable plaque formation.

Atherosclerotic plaque rupture is the main cause of acute coronary syndrome (ACS). Angiotensin II (Ang II) and macrophage apoptosis are involved in the pathogenesis of atherosclerosis. However, the underlying mechanisms remain unclear. We aimed to address the role of monocyte chemotactic protein-induced protein 1 (MCPIP1) in Ang II-induced macrophage apoptosis and vulnerable plaque formation. In mouse peritoneal macrophages, Ang II promoted endoplasmic reticulum (ER) stress-dependent macrophage apoptosis. Ang II markedly upregulated the expression of MCPIP1 via activating p38 mitogen-activated protein kinase (p38 MAPK). Treatment with MCPIP1 shRNA downregulated ER stress-related proteins and decreased macrophage apoptosis induced by Ang II. Ang II also activated the AMP-activated protein kinase (AMPK) signaling in macrophages. Inhibition of AMPK reduced macrophage apoptosis by inhibiting the p38 MAPK/MCPIP1/ER stress pathway. Furthermore, blocking the Ang II type 1 receptor (AT1R) with losartan effectively inhibited Ang II-induced macrophage apoptosis and AMPK/p38 MAPK/MCPIP1/ER pathway activation. In the atherosclerotic vulnerable plaque model in mice, losartan inhibited the progression of atherosclerosis and transformed vulnerable plaque into a more stable phenotype. Moreover, losartan markedly decreased the number of CD68+TUNEL+, CD68+MCPIP1+, CD68+p-eIF2α+ and CD68+CHOP+ cells in the lesion area. Taken together, Ang II promotes macrophage apoptosis via the AMPK/p38 MAPK/MCPIP1/ER stress pathway in macrophages via its receptor AT1R, which may contribute to vulnerable plaque formation. Our study clarifies a novel regulatory role of MCPIP1 in Ang II-induced macrophage apoptosis and plaque instability, providing a potential therapeutic target for prevention of ACS.

Structural Tailoring of a Novel Fluorescent IRE-1 RNase Inhibitor to Precisely Control Its Activity.

Activation of the IRE-1/XBP-1 pathway has been linked to many human diseases. We report a novel fluorescent tricyclic chromenone inhibitor, D-F07, in which we incorporated a 9-methoxy group onto the chromenone core to enhance its potency and masked the aldehyde to achieve long-term efficacy. Protection of the aldehyde as a 1,3-dioxane acetal led to strong fluorescence emitted by the coumarin chromophore, enabling D-F07 to be tracked inside the cell. We installed a photolabile structural cage on the hydroxy group of D-F07 to generate PC-D-F07. Such a modification significantly stabilized the 1,3-dioxane acetal protecting group, allowing for specific stimulus-mediated control of inhibitory activity. Upon photoactivation, the re-exposed hydroxy group on D-F07 triggered the aldehyde-protecting 1,3-dioxane acetal to slowly decompose, leading to the inhibition of the RNase activity of IRE-1. Our novel findings will also allow for spatiotemporal control of the inhibitory effect of other salicylaldehyde-based compounds currently in development.

How to Win the Battle with RNase.

Because ribose residues carry hydroxyl groups in both the 2' and 3' positions, RNA is chemically much more reactive than DNA and is easy prey to cleavage by contaminating RNases-enzymes with various specificities that share the property of hydrolyzing diester bonds linking phosphate and ribose residues. Because RNases are released from cells following lysis and are present on the skin, constant vigilance is required to prevent contamination of glassware and bench tops and the creation of aerosols carrying RNase. The problem is compounded because there is no simple method to inactivate RNases. Because of the presence of intrachain disulfide bonds, many RNases are resistant to prolonged boiling and mild denaturants and are able to refold quickly when denatured. Unlike many DNases, RNases do not require divalent cations for activity and thus cannot be easily inactivated by the inclusion of ethylenediaminetetraacetic acid (EDTA) or other metal ion chelators in buffer solutions. The best way to prevent problems with RNase is to avoid contamination in the first place.

A Human Ribonuclease Variant and ERK-Pathway Inhibitors Exhibit Highly Synergistic Toxicity for Cancer Cells.

Pancreatic-type ribonucleases (ptRNases) are prevalent secretory enzymes that catalyze the cleavage of RNA. Ribonuclease inhibitor (RI) is a cytosolic protein that has femtomolar affinity for ptRNases, affording protection from the toxic catalytic activity of ptRNases, which can invade human cells. A human ptRNase variant that is resistant to inhibition by RI is a cytotoxin that is undergoing a clinical trial as a cancer chemotherapeutic agent. We find that the ptRNase and protein kinases in the ERK pathway exhibit strongly synergistic toxicity toward lung cancer cells (including a KRASG12C variant) and melanoma cells (including BRAFV600E variants). The synergism arises from inhibiting the phosphorylation of RI and thereby diminishing its affinity for the ptRNase. These findings link seemingly unrelated cellular processes, and suggest that the use of a kinase inhibitor to unleash a cytotoxic enzyme could lead to beneficial manifestations in the clinic.

PEGylated DC-Chol/DOPE cationic liposomes containing KSP siRNA as a systemic siRNA delivery Carrier for ovarian cancer therapy.

Although siRNA-mediated downregulation technology has been highly successful in suppressing the expression of any disease-related gene, systemic delivery of siRNA for the clinical applications remains challenging, especially in the use of cancer therapy. DC-Chol/DOPE cationic liposomes as one of the most attractive vehicles for gene delivery have been widely exploited for transfection of siRNA into cells, but complexity of systemic delivery has allowed only their direct injection into local targets due to the formation of aggregations with negatively-charged blood components. Herein, we demonstrate the effects of PEGylation on DC-Chol/DOPE cationic liposomes for systemic siRNA delivery in cancer therapy. In contrast to non-PEGylated DC-Chol/DOPE-siRNA lipoplexes, PEGylated DC-Chol/DOPE-siRNA lipoplexes reduce the excretion by kidneys and scavenging in liver, prolonging the circulation time in vivo, and ultimately increase their preferential tumor accumulation. Therefore, systemic injection of PEGylated DC-Chol/DOPE liposomes loaded with siRNA against kinesin spindle protein (KSP) gene exhibited a high level of target gene silencing at tumor sites and substantial suppression of tumor growth. Furthermore, systemically administered PEGylated lipoplexes did not lead to any activation of innate immune responses in the immunocompetent mice. These results suggest the potential of PEGylated DC-Chol/DOPE liposomes as a systemic delivery carrier for siRNA-mediated cancer therapy.

Molecularly imprinted nanoparticles for inhibiting ribonuclease in reverse transcriptase polymerase chain reaction.

Molecularly imprinted nanoparticles (nanoMIPs) are synthesized via a solid-phase approach using RNase as the template. The feasibility of employing the nanoMIPs as RNase inhibitor is successfully demonstrated in reverse transcriptase polymerase chain reaction (RT-PCR) assays, suggesting the tailor-made nanomaterials are very promising for use in routine biological assays.

Antibody fusion proteins with human ribonucleases 1 to 8.

ImmunoRNases combine tumor targeting by antibodies with the cytotoxic action of ribonucleases from the RNase A superfamily. This study investigated for the first time all catalytic active human RNase A family members (1 to 8) as effector components of antibody fusion proteins. ImmunoRNase fusion proteins were constructed using the CD30-specific bivalent recombinant scFv-Fc antibody SH313-B5. Production of the resulting entirely human immunoRNases 1 to 8 was done in mammalian cells by secretion of active forms. The immunoRNases mediated CD30-specific cell binding and showed ribonucleolytic activity. Interestingly, immunoRNases 1 and 2 were active in the presence of up to 5-/20-fold molar excess of the pancreatic RNase inhibitor (RI), which is supposed to efficiently inhibit all human RNase A activity. ImmunoRNases 3, 4, 6 and 7 were only inhibited by several fold molar excess of RI, whereas immunoRNases 5 and 8 were already completely inactive at equimolar RI concentrations. Compared to free RNases, activity and RI sensitivity were not significantly changed by antibody fusion or dimerisation. ImmunoRNase3 and 5 mediated tumor growth inhibition at low nanomolar concentrations. Anti-tumor activity was antigen-specific and did not show any correlation with ribonucleolytic activity or RI sensitivity.

The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant.

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic ß-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes.

Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation.

The effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.

Allosteric mechanism of quinoline inhibitors for HIV RT-associated RNase with MD simulation and dynamics fluctuation network.

The human immunodeficiency virus (HIV) is a retrovirus which infects T lymphocyte of human body and causes immunodeficiency. Reverse transcriptase inhibitors (RTIs) can inhibit some functions of RT, preventing virus synthesis (double-stranded DNA), so that HIV virus replication can be reduced. Experimental results indicate a series of benzimidazole-based inhibitors which target HIV RT-associated RNase to inhibit the reverse transcription of HIV virus. However, the allosteric mechanism is still unclear. Here, molecular dynamics simulations and dynamics fluctuation network analysis were used to reveal the binding mode between the inhibitors and RT-associated RNase. The most active molecule has more hydrophobic and electrostatic interactions than the less active inhibitor. Dynamics correlation network analysis indicates that the most active inhibitor perturbs the network of RT-associated RNase and decreases the correlation of nodes. 3D-QSAR model suggests that two robust and reliable models were constructed and validated by independent test set. 3D-QSAR model also shows that bulky negatively charged or hydrophilic substituent is favorable to bioactivity. These results reveal the allosteric mechanism of quinoline inhibitors and help to improve the bioactivity.