Degradation Of Ribonucleic Acid Research Articles

The potential of RNA therapeutics in dermatology.

Ribonucleic acid (RNA) therapeutics hold great potential for the advancement of dermatological treatments due to, among other reasons, the possibility of treating previously undruggable targets, high specificity with minimal side effects, and ability to include multiple RNA targets in a single product. Although there have been research relating to RNA therapeutics for decades, there have not been many products translated for clinical use until recently. This may be because of challenges to the application of RNA therapeutics, including the dearth of effective modes of delivery to the target, and rapid degradation of RNA in the human body and environment. This article aims to provide insight on (1) the wide-ranging possibilities of RNA therapeutics in the field of dermatology as well as (2) how key challenges can be addressed, so as to encourage the development of novel dermatological treatments. We also share our experience on how RNA therapeutics have been applied in the management of hypertrophic and keloid scars.

Degradation of SARS-CoV-2 specific ribonucleic acid in samples for nucleic acid amplification detection

The degradation of SARS-CoV-2 specific ribonucleic acid (RNA) was investigated by a numerical modeling approach based on nucleic acid amplification test (NAAT) results utilizing the SmartAmp technique. The precision of the measurement was verified by the relative standard deviation (RSD) of repeated measurements at each calibration point. The precision and detection limits were found to be 6% RSD (seven repeated measurements) and 94 copies/tube, respectively, at the lowest calibration point. RNA degradation curves obtained from NAAT data on four different temperatures were in good agreement with the first-order reaction model. By referring to rate constants derived from the results, the Arrhenius model was applied to predict RNA degradation behavior. If the initial RNA concentration was high enough, such as in samples taken from infected bodies, the NAAT results were expected to be positive during testing. On the other hand, if initial RNA concentrations were relatively low, such as RNA in residual viruses on environmental surfaces, special attention should be paid to avoid false-negative results. The results obtained in this study provide a practical guide for RNA sample management in the NAAT of non-human samples.

Identification of an eight-m6A RNA methylation regulator prognostic signature of uterine corpus endometrial carcinoma based on bioinformatics analysis.

N6-methyladenosine (m6A) methylation is proved to play a significant role in human cancers. This study aimed to explore the association between m6A ribonucleic acid (RNA) methylation regulators and uterine corpus endometrial carcinoma (UCEC), and build a prognostic signature of m6A regulators for UCEC.RNA-seq transcriptome data and clinicopathological data of UCEC were downloaded from the Cancer Genome Atlas database. We compared the expression of 23 m6A-regulators in tumor tissues and nontumor tissues. Then we classified the data into 3 clusters by consensus clustering analysis. Several regulators were picked out as the prognostic signature of patients with UCEC based on least absolute shrinkage and selection operator Cox regression analysis. Additionally, we established a predictive nomogram to calculate survival times. Finally, we used receiver operating characteristic curve, univariate Cox regression analysis, and multivariate Cox regression analysis to further verify the prognostic value of the risk signature consisting of m6A regulators.The expression of 18/23 m6A regulators was significantly different in UCEC compared with normal samples. Gene ontology functional analysis of these regulators revealed that they were mainly participated in RNA splicing, stabilization, modification, and degradation. LRPPRC, IGFBP2, KIAA1429, IGFBP3, FMR1, YTHDF1, METTL14, and YTHDF2 were selected to construct the risk signature and predictive nomogram. The results of receiver operating characteristic curve, univariate Cox regression analysis, and multivariate Cox regression analysis for the risk signature showed a good predictive performance for UCEC.The risk signature of 8-m6A regulators has potential prognostic value for patients with UCEC.

Nanoparticle mediated RNA delivery for wound healing.

Wound healing is a complicated physiological process that comprises various steps, including hemostasis, inflammation, proliferation, and remodeling. The wound healing process is significantly affected by coexisting disease states such as diabetes, immunosuppression, or vascular disease. It can also be impacted by age, repeated injury, or hypertrophic scarring. These comorbidities can affect the rate of wound closure, the quality of wound closure, and tissues' function at the affected sites. There are limited options to improve the rate or quality of wound healing, creating a significant unmet need. Advances in nucleic acid research and the human genome project have developed potential novel approaches to address these outstanding requirements. In particular, the use of microRNA, short hairpin RNA, and silencing RNA is unique in their abilities as key regulators within the physiologic machinery of the cell. Although this innovative therapeutic approach using ribonucleic acid (RNA) is an attractive approach, the application as a therapeutic remains a challenge due to site-specific delivery, off-target effects, and RNA degradation obstacles. An ideal delivery system is essential for successful gene delivery. An ideal delivery system should result in high bioactivity, inhibit rapid dilution, controlled release, allow specific activation timings facilitating physiological stability, and minimize multiple dosages. Currently, these goals can be achieved by inorganic nanoparticle (NP) (e.g., cerium oxide, gold, silica, etc.) based delivery systems. This review focuses on providing insight into the preeminent research carried out on various RNAs and their delivery through NPs for effective wound healing. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Recent trends in application of nanomaterials for the development of electrochemical microRNA biosensors.

The biology of the late twentieth century was marked by the discovery in 1993 of a new class of small non-coding ribonucleic acids (RNAs) which play major roles in regulating the translation and degradation of messenger RNAs. These small RNAs (18-25 nucleotides), called microRNAs (miRNAs), are implied in several biological processes such as differentiation, metabolic homeostasis, or cellular apoptosis and proliferation. The discovery in 2008 that the presence of miRNAs in body fluids could be correlated with cancer (prostate, breast, colon, lung, etc.) or other diseases (diabetes, heart diseases, etc.) has made them new key players as biomarkers. Therefore, miRNA detection is of considerable significance in both disease diagnosis and in the study of miRNA function. Until these days, more than 1200 miRNAs have been identified. However, traditional methods developed for conventional DNA does not apply satisfactorily for miRNA, in particular due to the low expression level of these miRNA in biofluids, and because they are very short strands. Electrochemical biosensors can provide this sensitivity and also offer the advantages of mass fabrication, low-cost, and potential decentralized analysis, which has wide application for microRNAs sensing, with many promising results already reported. The present reviewsummarizessome newly developed electrochemical miRNA detection methods.

Metabolomics combined with proteomics provides a novel interpretation of the changes in nonvolatile compounds during white tea processing.

In this study, metabolomics and proteomics were employed to investigate the change mechanism of nonvolatile compounds during white tea processing. A total of 99 nonvolatile compounds were identified, among which the contents of 13 free amino acids, caffeine, theaflavins, 7 nucleosides and nucleotides, and 5 flavone glycosides increased significantly, while the contents of theanine, catechins, theasinesins, 3 proanthocyanidins, and phenolic acids decreased significantly during the withering period. The results of proteomics indicated that the degradation of proteins accounted for the increase in free amino acid levels; the weakened biosynthesis, in addition to oxidation, also contributed to the decrease in flavonoid levels; the degradation of ribonucleic acids contributed to the increase in nucleoside and nucleotide levels during the withering period. In addition, the drying process was found to slightly promote the formation of white tea taste. Our study provides a novel characterization of white tea taste formation during processing.

Semiconducting Polymer Nanomaterials as Near-Infrared Photoactivatable Protherapeutics for Cancer.

Cancer therapy is routinely performed in the clinic to cure cancer and control its progression, wherein therapeutic agents are generally used. To reduce side effects, protherapeutic agents that can be activated by overexpressed cancer biomarkers are under development. However, these agents still face certain extent of off-target activation in normal tissues, stimulating the interest to design external-stimuli activatable protherapeutics. In this regard, photoactivatable protherapeutic agents have been utilized for cancer treatments. However, because of the intrinsic features of photolabile moieties, most photoactivatable protherapeutic agents only respond to ultraviolet-visible light, limiting their in vivo applications. Thus, protherapeutic agents that can be activated by near-infrared (NIR) light with minimal phototoxicity and increased tissue penetration are highly desired.In this Account, we summarize our semiconducting polymer nanomaterials (SPNs) as NIR photoactivatable protherapeutic agents for cancer treatment. SPNs are transformed from π-conjugated polymers that efficiently convert NIR light into heat or singlet oxygen (1O2). With photothermal and photodynamic properties, SPNs can be directly used as photomedicine or serve as light transducers to activate heat or 1O2-responsive protherapeutic agents.The heat-activatable SPN-based protherapeutic agents are developed by loading or conjugating of SPNs with therapeutic agents (e.g., agonist, gene, and enzyme). For instance, photothermally triggered release of agonists specifically activates certain protein ion channels on the cellular membrane, leading to ion overinflux induced mitochondria dysfunction and consequently apoptosis of cancer cells. Moreover, photothermal activation of temperature-sensitive bromelain can promote the in situ degradation of collagens (the major components of extracellular matrix), resulting in an improved accumulation of agents in tumor tissues and thus amplified therapeutic outcome.The 1O2-activatable SPN-based protherapeutic agents are constructed through covalent conjugation of SPNs with caged therapeutic agents via hypoxia- or 1O2-cleavable linkers. Upon NIR photoirradiation, SPNs consume oxygen to generate 1O2, which leads to photodynamic therapy (PDT), and meanwhile breaks hypoxia- or 1O2-cleavable linkers for on-demand release and in situ activation of caged protherapeutic molecules (e.g., chemodrug, enzyme, and inhibitor). Such remote activation of SPN-based protherapeutic agents can be applied to induce DNA damage, ribonucleic acid degradation, inhibition of protein biosynthesis, or immune system activation in tumors of living animals. By synergizing PDT with NIR photoactivation of those biological actions, these protherapeutic agents effectively eliminate tumors and even fully inhibit tumor metastasis.This Account highlights the potential of SPNs for construction of versatile NIR photoactivatable protherapeutics to treat cancer at designated times and locations with high therapeutic outcome and precision.

Estimating time since deposition using quantification of RNA degradation in body fluid-specific markers

The first appearance of ribonucleic acid (RNA) in forensic science research was in 1984 in the study of post-mortem tissues. Since then, many studies have explored the role of gene expression and its potential applications in forensic science. The two main RNA molecules that have been subject to increasing interest in the forensic science community are messenger RNA (mRNA) and microRNA (miRNA). Identification of body fluid type and estimating the time since deposition can be of immense value to criminal investigations. Determining the time since deposition or age of a biological stain can help to indicate either when a crime happened, or whether the biological evidence was deposited before/after a known crime event, in order for samples to be excluded. The research presented here has used reverse transcription quantitative PCR to examine the relative expression ratio (RER) in two types of body fluid-specific markers (saliva and semen), to develop a method to estimate the age of biological stains. mRNA and miRNA markers specific to saliva and semen, along with three reference genes were selected. Biological samples from 20 participants were stored in a dark dry place at room temperature to simulate natural ageing. A series of desired ageing points were set and total RNA was extracted when samples reached each desired point. The degradation behaviour of each RNA marker was analysed, showing that they exhibited unique degradation profiles across a one-year storage interval for saliva and semen samples, where miRNAs and the U6 reference gene were shown to have high stability. The RERs exhibit a non-linear relationship with body fluid stain age and can be considered as a potential method for body fluid stain age estimation, hence the time since deposition.

Photoactivatable Organic Semiconducting Pro-nanoenzymes.

Therapeutic enzymes hold great promise for cancer therapy; however, in vivo remote control of enzymatic activity to improve their therapeutic specificity remains challenging. This study reports the development of an organic semiconducting pro-nanoenzyme (OSPE) with a photoactivatable feature for metastasis-inhibited cancer therapy. Upon near-infrared (NIR) light irradiation, this pro-nanoenzyme not only generates cytotoxic singlet oxygen (1O2) for photodynamic therapy (PDT), but also triggers a spontaneous cascade reaction to induce the degradation of ribonucleic acid (RNA) specifically in tumor microenvironment. More importantly, OSPE-mediated RNA degradation is found to downregulate the expression of metastasis-related proteins, contributing to the inhibition of metastasis after treatment. Such a photoactivated and cancer-specific synergistic therapeutic action of OSPE enables complete inhibition of tumor growth and lung metastasis in mouse xenograft model, which is not possible for the counterpart PDT nanoagent. Thus, our study proposes a phototherapeutic-proenzyme approach toward complete-remission cancer therapy.

RNA Degradation in Neurodegenerative Disease.

Ribonucleic acid (RNA) homeostasis is dynamically modulated in response to changing physiological conditions. Tight regulation of RNA abundance through both transcription and degradation determines the amount, timing, and location of protein translation. This balance is of particular importance in neurons, which are among the most metabolically active and morphologically complex cells in the body. As a result, any disruptions in RNA degradation can have dramatic consequences for neuronal health. In this chapter, we will first discuss mechanisms of RNA stabilization and decay. We will then explore how the disruption of these pathways can lead to neurodegenerative disease.

Tabu Search for the RNA Partial Degradation Problem

Abstract In recent years, a growing interest has been observed in research on RNA (ribonucleic acid), primarily due to the discovery of the role of RNA molecules in biological systems. They not only serve as templates in protein synthesis or as adapters in the translation process, but also influence and are involved in the regulation of gene expression. The RNA degradation process is now heavily studied as a potential source of such riboregulators. In this paper, we consider the so-called RNA partial degradation problem (RNA PDP). By solving this combinatorial problem, one can reconstruct a given RNA molecule, having as input the results of the biochemical analysis of its degradation, which possibly contain errors (false negatives or false positives). From the computational point of view the RNA PDP is strongly NP-hard. Hence, there is a need for developing algorithms that construct good suboptimal solutions. We propose a heuristic approach, in which two tabu search algorithms cooperate, in order to reconstruct an RNA molecule. Computational tests clearly demonstrate that the proposed approach fits well the biological problem and allows to achieve near-optimal results. The algorithm is freely available at http://www.cs.put.poznan.pl/arybarczyk/tabusearch.php.

Purification and characterization of an extracellular ribonuclease from a Bacillus sp. RNS3 (KX966412)

Ribonucleases (RNases) catalyze the degradation of ribonucleic acid (RNA) into smaller nucleotides. RNases display angiogenic, neurotoxic, antitumor and immunosuppressive properties. In the present study, an extracellular RNase was successfully purified to homogeneity from a Bacillus sp. RNS3 (KX966412) by salting out at 0–50% ammonium sulphate saturation followed by the gel permeation (Sephadex G-100) chromatography. The multistep purification resulted in 10.4 fold purification of RNase with a yield of 3.12%. The activity of the purified RNase was found to be 2.02U/mg protein. The purified RNase was monomeric with a molecular weight of 66kDa. It exhibited Michalis-Menten kinetics parameters Kcat 7.92min−1 and Km 0.12mg/mL. The antiproliferative activity of the purified RNase was tested against an established Hep-2C (HeLa derived) cancer cell line in vitro. The purified RNase reduced the viability of the Hep-2C cells significantly with an IC50 value of 3.53μg/mL. The haemolytic activity of purified RNase was also evaluated and unfortunately, it showed a strong haemolytic activity towards human RBCs.

Ribonuclease as Anticancer Therapeutics

Ribonucleases (RNases) are small molecules which are highly cytotoxic in nature. They catalyse the degradation of RNA into smaller molecules rapidly in an unprotected environment. The cytotoxic properties of RNases include degradation of RNA leading to blockage of protein synthesis in malignant cells and inducing the apoptosis response. Cytotoxicity of RNases is determined by catalytic activity, stability, non-selective nature of inhibitors, positive charge on molecule and internalization. Onconase, BS-RNase and other RNases exert cytotoxic activity on cancer cells selectively by involving different cellular pathways and/ or enhance the cytotoxicity by mutation. A general mechanism of the cytotoxic activity of RNases includes the interaction of the enzyme with the cellular membrane by non-specific interactions mediated by Coulombic forces, internalization by endocytosis, translocation to the cytosol, degradation of ribonucleic acid and subsequent cell death by activation of caspase-dependent mechanisms, low molecular weight compounds or alteration in protein and NF-κB signal pathway. But still it is unclear that which of these mechanisms is most potent, common and causes cell death in cancerous cells. The problem related to ribonuclease inhibitor (RI) has not fully elucidated. This article looks at the cellular pathways of RNases and mechanism of their cytotoxicity towards the malignant cells which makes RNase as a strong candidate to be considered as chemotherapeutic or antitumor drug. Some of the prominent approaches to exploit RNase molecule as an anticancer therapeutic agent have been discussed.

A Novel Method to Quantify RNA-Protein Interactions In Situ Using FMTRIP and Proximity Ligation.

RNA binding proteins (RBP) and small RNAs regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNAs) through their interactions with specific cis-acting elements within target RNAs. Here, we describe a novel method to detect protein-mRNA interactions, which combines FLAG-peptide modified, multiply-labeled tetravalent RNA imaging probes (FMTRIPs) with proximity ligation (PLA), and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity.

Effect of Temperature towards RNA Concentration: Quantitative Investigation with Spectrophotometer

Ribonucleic acid (RNA) is a thermodynamically unstable molecule. The way RNA samples are preserved is critical to maintain maximum yield and quality, therefore it is useful for molecular analysis such as real time- PCR. There are many contradictions and variations regarding the temperature for RNA storage. The aim of this study is to find the ideal temperature to store RNA among -80o C, -20o C and 4oC by determining changes in RNA concentration over two weeks of storage time. The liver of eight rats was divided into three groups, weighing from 25-26 μg. Samples were homogenized, isolated and stored in -80oC, -20oC and 4oC freezers. Absorbance was measured with spectrophotometry at 260 and 280 nm to determine the concentration of RNA. There was no significant difference in the concentration of RNA samples stored in all temperatures after two weeks, both experimentally and statistically (Kruskal-Wallis, -80oC p=0.949; -20oC p=0.885; 4oC p=0.935). In conclusion, RNA can be stored in -80oC, -20oC and 4oC for two weeks without quantity reduction. Longer duration of study and RNA quality analysis is recommended to check for RNA degradation. Normal 0 false false false IN X-NONE X-NONE

RNA extraction for RNA sequencing of archival renal tissues

Background: Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS.Method: We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq® RNA Access Library Preparation Kit.Conclusion: Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.

Can we observe changes in mRNA "state"? Overview of methods to study mRNA interactions with regulatory proteins relevant in cancer related processes.

RNA binding proteins (RBP) regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNA) through their interactions with specific cis-acting elements within target RNAs. Post-transcriptional regulatory mechanisms are directly involved in the control of the immune response and stress response and their alterations play a crucial role in cancer related processes. In this review, we discuss mRNAs and RNA binding proteins relevant to tumorigenesis, current methodologies for detecting RNA interactions, and last, we describe a novel method to detect such interactions, which combines peptide modified, RNA imaging probes (FMTRIPs) with proximity ligation (PLA) and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity in situ.

Current perspectives on the role of TRAMP in nuclear RNA surveillance and quality control

The TRAMP complex assists the nuclear exosome to degrade a broad range of ribonucleic acid (RNA) substrates by increasing both exoribonucleolytic activity and substrate specificity. However, how the interactions between the TRAMP subunits and the components of the nuclear exosome regulate their functions in RNA degradation and substrate specificity remain unclear. This review aims to provide a summary of the recent findings on the role of the TRAMP complex in nuclear RNA degradation. The new insights from recent structural biological studies are discussed.

Internalization of Bacillus intermedius ribonuclease (BINASE) induces human alveolar adenocarcinoma cell death

Ribonuclease (RNase) treatment represents a novel mechanism based approach to anticancer therapy as an alternative to the DNA damaging drugs commonly used in clinical practice. Apart from their ribonucleolytic activity, cytotoxic effects have attracted a considerable attention to RNases because of their potential as selective agents for treatment of certain malignancies. Among these enzymes, Binase, an RNase from Bacillus intermedius, has shown promising results. Here, we have found that binase selectively attacked human A549 alveolar adenocarcinoma cells to trigger an apoptotic response, whereas normal lung epithelial cells LEK were not affected by the ribonuclease. The tumor transformation led to the modification of certain cellular characteristics causing cell sensitivity to binase. Although a general mode for RNases cytotoxicity includes their penetration into the cell, translocation to the cytosol and degradation of ribonucleic acid, many aspects of this process have not been fully elucidated. Our data revealed the following time-dependent changes induced by binase in A549 cells: (a) fast permanent internalization of the enzyme during the first hours of treatment; (b) temporary increase in cellular permeability for macromolecules during the 4–6 h of treatment; (c) apoptotic alterations in population after 24 h and (d) DNA fragmentation and cell death after 72 h of treatment with binase. Elucidation of these molecular strategies used by this promising toxin provides us essential information for the development of new anticancer drugs.

Antibody detection of translocations in Ewing sarcoma

The detection of chromosomal translocations has important implications in the diagnosis, prognosis and treatment of patients with cancer. Current approaches to translocation detection have significant shortcomings, including limited sensitivity and/or specificity, and difficulty in application to formalin-fixed paraffin-embedded (FFPE) clinical samples. We developed a new approach called antibody detection of translocations (ADOT) that avoids the shortcomings of current techniques. ADOT combines a transcriptional microarray-based approach with a novel antibody-based detection method. ADOT allows for the accurate and sensitive identification of translocations and provides exon-level information about the fusion transcript. ADOT can detect translocations in poor-quality unprocessed total ribonucleic acid (RNA). Furthermore, the technique is readily generalizable to detect any potential fusion transcript, including previously undescribed fusions. We demonstrate the feasibility of ADOT by examples in which both known and unknown Ewing sarcoma translocations are identified from cell lines, tumour xenografts and FFPE primary tumours. These results demonstrate that ADOT may be an effective approach for translocation analysis in clinical specimens with significant RNA degradation and may offer a novel diagnostic tool for translocation-based cancers.