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Rnase h

Peter H Hagedorn, Malene Pontoppidan, Tina S Bisgaard, Marco Berrera, Andreas Dieckmann, Martin Ebeling, Marianne R Møller, Heidi Hudlebusch, Marianne L Jensen, Henrik F Hansen, Troels Koch, Morten Lindow
Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets...
May 22, 2018: Nucleic Acids Research
Mohammad Gholami, Mehrdad Ravanshad, Kazem Baesi, Siamak M. Samiee, Negin Hosseini Rozbahani, Minoo Mohraz
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...
May 19, 2018: Iranian Biomedical Journal
Hirokazu Takahashi, Masahiko Ohkawachi, Kyohei Horio, Toshiro Kobori, Tsunehiro Aki, Yukihiko Matsumura, Yutaka Nakashimada, Yoshiko Okamura
RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes...
May 17, 2018: Scientific Reports
Yuchen Nan, Yan-Jin Zhang
Phosphorodiamidate morpholino oligomers (PMO) are short single-stranded DNA analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. As uncharged nucleic acid analogs, PMO bind to complementary sequences of target mRNA by Watson-Crick base pairing to block protein translation through steric blockade. PMO interference of viral protein translation operates independently of RNase H. Meanwhile, PMO are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications...
2018: Frontiers in Microbiology
Jianqing Chen, Peng Wang, Barend H J de Graaf, Hao Zhang, Huijun Jiao, Chao Tang, Shaoling Zhang, Juyou Wu
S-RNase is the female determinant of self-incompatibility (SI) in pear (Pyrus bretschneideri). After translocation to the pollen tube, S-RNase degrades rRNA and induces pollen tube death in an S-haplotype-specific manner. In this study, we found that the actin cytoskeleton is a target of P. bretschneideri S-RNase (PbrS-RNase) and uncovered a mechanism that involves phosphatidic acid (PA) and protects the pollen tube from PbrS-RNase cytotoxicity. PbrS-RNase interacts directly with PbrActin1 in an S-haplotype-independent manner, causing the actin cytoskeleton to depolymerize and promoting programmed cell death in the self-incompatible pollen tube...
April 30, 2018: Plant Cell
Kevin P Larsen, Yamuna Kalyani Mathiharan, Kalli Kappel, Aaron T Coey, Dong-Hua Chen, Daniel Barrero, Lauren Madigan, Joseph D Puglisi, Georgios Skiniotis, Elisabetta Viani Puglisi
Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection 1 and a common target of antiretroviral drugs 2 . The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA 5 each bound to host lysine 3 transfer RNA (tRNALys 3 ), which acts as a primer for initiation of reverse transcription6,7 . Upon viral entry into cells, initiation is slow and non-processive compared to elongation8,9 ...
April 25, 2018: Nature
Cassandra Herbert, Yannick Kokouvi Dzowo, Anthony Urban, Courtney N Kiggins, Marino J E Resendiz
Understanding how oxidatively damaged RNA interacts with ribonucleases is of importance due to its proposed role in the development/progression of disease. Thus, understanding structural aspects of RNA containing lesions generated under oxidative stress, as well as its interactions with other biopolymers is fundamental. We explored the reactivity of RNase A, RNase T1, and RNase H towards oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful towards understanding 1) how these RNases can be used to characterize the structural impact that this lesion has on RNA; and 2) how oxidatively modified RNA may be handled intracellularly...
April 23, 2018: Biochemistry
Yajun Wang, Erkai Liu, Curtis H Lam, David M Perrin
Sequence-specific cleavage of RNA targets in the absence of a divalent metal cation (M2+ ) has been a long-standing goal in bioorganic chemistry. Herein, we report the in vitro selection of novel RNA cleaving DNAzymes that are selected using 8-histaminyl-deoxyadenosine (dAim TP), 5-guanidinoallyl-deoxyuridine (dUga TP), and 5-aminoallyl-deoxycytidine (dCaa TP) along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A. Of several such M2+ -free DNAymes, DNAzyme 7-38-32 cleaves a 19 nt all-RNA substrate with multiple-turnover, under simulated physiological conditions wherein only 0...
February 21, 2018: Chemical Science
Albert Yen, Yilong Cheng, Meilyn Sylvestre, Heather Herd Gustafson, Sanyogitta Puri, Suzie H Pun
Messenger RNA (mRNA) is a biomolecule with a wide range of promising clinical applications. However, the unstable nature of mRNA and its susceptibility to degradation by ribonucleases necessitate the use of specialized formulations for delivery. Polycations are an emerging class of synthetic carriers capable of packaging nucleic acids, and may serve as suitable ribonuclease-resistant formulations for mRNA administration. Here, we explore the application of VIPER and sunflower polycations, two polycations previously synthesized by our group, for the delivery of mRNA in comparison to branched poly(ethylenimine); all three polycations have been shown to efficiently deliver plasmid DNA (pDNA) to cultured cells...
April 19, 2018: Molecular Pharmaceutics
Elbashir Araud, Joanna L Shisler, Thanh H Nguyen
Two rotavirus (RV) strains (sialidase-resistant Wa and sialidase-sensitive OSU) were irradiated with simulated solar UVA and visible light in sensitizer-free phosphate buffered solution (lacking exogenous reactive oxygen species (ROS)) or secondary effluent wastewater (producing ROS). Although light attenuated for up to 15% through the secondary effluent wastewater (SEW), the inactivation efficacies increased by 0.7 log10 for Wa and 2 log10 for OSU compared to those in sensitizer-free phosphate buffered solution (PBS) after 4 hrs of irradiation...
April 19, 2018: Environmental Science & Technology
Youlong Ma, Feiyue Teng, Matthew R Libera
Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here we demonstrate the concept of solid-phase nucleic acid sequence based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency...
April 13, 2018: Analytical Chemistry
Melissa Drappier, Babal Kant Jha, Sasha Stone, Ruth Elliott, Rong Zhang, Didier Vertommen, Susan R Weiss, Robert H Silverman, Thomas Michiels
The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler's murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L...
April 2018: PLoS Pathogens
Jennifer J Wanat, Glennis A Logsdon, Jordan H Driskill, Zhong Deng, Paul M Lieberman, F Brad Johnson
The events underlying senescence induced by critical telomere shortening are not fully understood. Here we provide evidence that TERRA, a non-coding RNA transcribed from subtelomeres, contributes to senescence in yeast lacking telomerase (tlc1Δ). Levels of TERRA expressed from multiple telomere ends appear elevated at senescence, and expression of an artificial RNA complementary to TERRA (anti-TERRA) binds TERRA in vivo and delays senescence. Anti-TERRA acts independently from several other mechanisms known to delay senescence, including those elicited by deletions of EXO1, TEL1, SAS2, and genes encoding RNase H enzymes...
2018: PloS One
Paul L Boyer, Steven J Smith, Xue Zhi Zhao, Kalyan Das, Kevin Gruber, Eddy Arnold, Terrence R Burke, Stephen H Hughes
We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA/DNA heteroduplex. The compounds were tested using a variety of substrates: All three compounds inhibited the polymerase-independent RH activity of HIV-1 RT...
April 11, 2018: Journal of Virology
Yan Zhang, David H Burkhardt, Silvi Rouskin, Gene-Wei Li, Jonathan S Weissman, Carol A Gross
Temperature influences the structural and functional properties of cellular components, necessitating stress responses to restore homeostasis following temperature shift. Whereas the circuitry controlling the heat shock response is well understood, that controlling the E. coli cold shock adaptation program is not. We found that during the growth arrest phase (acclimation) that follows shift to low temperature, protein synthesis increases, and open reading frame (ORF)-wide mRNA secondary structure decreases...
April 2, 2018: Molecular Cell
Hongchang Zhao, Min Zhu, Oliver Limbo, Paul Russell
In Saccharomyces cerevisiae , genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA-DNA hybrids (R-loops). In Schizosaccharomyces pombe , RNase H enzymes were reported to process RNA-DNA hybrids produced at a double-strand break (DSB) generated by I-PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1∆ rnh201∆ cells, which lack the RNase H enzymes, accumulate R-loops and activate DNA damage checkpoints...
April 5, 2018: EMBO Reports
Alvaro Ringressi, Veronica Cunsolo, Francesca Malentacchi, Simone Pozzessere
BACKGROUND: The Bombay phenotype is a rare genetic trait which is characterized by the absence of A, B and H antigens on red cells as well as in body secretions. The serum shows the presence of antibodies against antigen H. Patients with this rare blood type are not easily transfusable. We had observed a woman aged 18, at the 20th week of pregnancy, native of Sri Lanka, with an IgG and IgM class anti-H. We report the case and the clinical issues arisen. MATERIALS AND METHODS: The determination of ABO, Rh[D] group, the indirect antiglobulin test (IAT) were performed in tube techniques and in neutral gel microcolumn...
January 2018: Annali Dell'Istituto Superiore di Sanità
S Palikša, G Alzbutas, R Skirgaila
Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display technique for in vitro evolution of the Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT) that would be able to perform efficient full-length cDNA synthesis in PCR buffer optimized for Thermus aquaticus DNA polymerase...
March 1, 2018: Protein Engineering, Design & Selection: PEDS
Ying-Nai Wang, Heng-Huan Lee, Chao-Kai Chou, Wen-Hao Yang, Yongkun Wei, Chun-Te Chen, Jun Yao, Jennifer L Hsu, Cihui Zhu, Haoqiang Ying, Yuanqing Ye, Wei-Jan Wang, Seung-Oe Lim, Weiya Xia, How-Wen Ko, Xiuping Liu, Chang-Gong Liu, Xifeng Wu, Huamin Wang, Donghui Li, Laura R Prakash, Matthew H Katz, Yaan Kang, Michael Kim, Jason B Fleming, David Fogelman, Milind Javle, Anirban Maitra, Mien-Chie Hung
Pancreatic ribonuclease (RNase) is a secreted enzyme critical for host defense. We discover an intrinsic RNase function, serving as a ligand for epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase (RTK), in pancreatic ductal adenocarcinoma (PDAC). The closely related bovine RNase A and human RNase 5 (angiogenin [ANG]) can trigger oncogenic transformation independently of their catalytic activities via direct association with EGFR. Notably, high plasma ANG level in PDAC patients is positively associated with response to EGFR inhibitor erlotinib treatment...
March 29, 2018: Cancer Cell
Cinzia Sanna, Monica Scognamiglio, Antonio Fiorentino, Angela Corona, Vittoria Graziani, Alessia Caredda, Pierluigi Cortis, Mariofilippo Montisci, Elisa Rita Ceresola, Filippo Canducci, Ferruccio Poli, Enzo Tramontano, Francesca Esposito
In a search for new potential multitarget anti-HIV compounds from natural products, we have identified in Hypericum scruglii, an endemic and exclusive species of Sardinia (Italy), a potent plant lead. The phytochemical study of the hydroalcoholic extract obtained from its leaves led to the isolation of its most abundant secondary metabolites, belonging to different chemical classes. In particular, three phloroglucinols derivatives were identified, confirming their significance as chemotaxonomic markers of the Hypericum genus...
2018: PloS One
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