Suchergebnisse

Upon the discovery of the RNA interference pathway, the development of nucleic acids derivatives for therapeutic purposes has soon caught the attention of biomedical researchers. Although synthetic small interfering RNA (siRNA) has been extensively used to downregulate any protein-coding mRNA, several key issues still remain unsolved. The acyclic threoninol nucleic acid (aTNA), placed at certain siRNA positions, is a useful modification to reduce the oligonucleotides vulnerability towards nucleases. In addition, it can be exploited to avoid several OFF-target effects that limit the biological safety of the RNAi-based agents. ; We thank the European Union (NMP4-LA-2011-262943, MULTIFUN), the Spanish MINECO (CTQ2014-52588-R and CTQ2014-61758-EXP), and the Generalitat de Catalunya for funding this research. CIBER-BBN is financed by the European Regional Development Fund and the Instituto de Salud Carlos III through an initiative funded during the VI Plan Nacional 2008-2011, the Ingenio 2010, the Consolider Program, and the CIBER Action. ; Peer reviewed

Hybrid Oncidium orchids, such as Oncidium Gower Ramsey and Oncidium "Honey Angel," are popular cut flowers in Japan and Taiwan. Due to pollen sterility, no new varieties have been created by conventional breeding methods. Recently, we employed RNA interference (RNAi) technology to suppress phytoene synthase and successfully modified floret hue from yellow to white (Liu et al. 2019). Transgenic white Oncidium orchids, Honey Snow MF-1, have been grown to test their genetic stability, and their environmental biosafety was assessed for approximately one year under government regulatory instructions from the Council of Agriculture, Taiwan. In the present study, pollen sterility was demonstrated by cytological observation of the microsporogenesis step, pollen morphology abortion, and failure of pollen germination. Assays on allelopathic effect on the other plants and the soil rhizospheric microbial flora-revealed that transgenic Oncidium orchids are potentially safe with regard to environmental biodiversity. Therefore, the general release permissions have been granted and an application for licensing for commercial production is under way.

The understanding of the mechanisms behind nucleotide recognition by Argonaute 2, core protein of the RNA-induced silencing complex, is a key aspect in the optimization of small interfering RNAs (siRNAs) activity. To date, great efforts have been focused on the modification of certain regions of siRNA, such as the 3′/5′-termini and the seed region. Only a few reports have described the roles of central positions flanking the cleavage site during the silence process. In this study, we investigate the potential correlations between the thermodynamic and silencing properties of siRNA molecules carrying, at internal positions, an acyclic L-threoninol nucleic acid (aTNA) modification. Depending on position, the silencing is weakened or impaired. Furthermore, we evaluate the contribution of mismatches facing either a natural nucleotide or an aTNA modification to the siRNA potency. The position 11 of the antisense strand is more permissive to mismatches and aTNA modification, in respect to the position 10. Additionally, comparing the ON-/OFF-target silencing of central mismatched siRNAs with 5′-terminal modified siRNA, we concluded: (i) central perturbation of duplex pairing features weights more on potency rather than silencing asymmetry; (ii) complete bias for the ON-target silencing can be achieved with single L-threoninol modification near the 5'-end of the sense strand. ; This study was supported by the European Union (MULTIFUN, NMP4-LA-2011-262943), the Spanish Ministry of Education (CTQ2010-20541), Generalitat de Catalunya (2009/SGR/208). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. We are indebted to Dr. Elisa Pedone for her helpful advice and for providing technical assistance. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed

North bicyclo methanocarba thymidine (TN) nucleosides were substituted into siRNAs to investigate the effect of bicyclo[3.1.0]hexane 2′-deoxy-pseudosugars on RNA interference activity. Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery. We studied the effect of the TN modification in a screen involving residue-specific changes in an siRNA targeting Renilla luciferase and we applied the most effective pattern of modification to the knockdown of murine tumor necrosis factor (TNF-α). We also showed that incorporation of TN units into siRNA duplexes increased their thermal stabilities, substantially enhanced serum stabilities, and decreased innate immunostimulation. Comparative RNAi studies involving the TN substitution and locked nucleic acids (LNAs) showed that the gene-silencing activities of TN-modified siRNAs were comparable to those obtained with the LNA modification. An advantage of the North 2′-deoxy-methanocarba modification is that it may be explored further in the future by changing the 2′-position. The results from these studies suggest that this modification might be valuable for the development of siRNAs for therapeutic applications. ; This research was supported by the European Union (MULTIFUN consortium), the Spanish Ministry of Education (BFU2007-63287, CTQ2010-20541), and the Generalitat the Catalunya (2009/SGR/208). This work was funded in part by the Center for Cancer Research, National Cancer Institute, NIH. M.T. acknowledges the JAE-Doc 2008 contract (CSIC, Spain) and the Juan de la Cierva contract (MICINN, Spain) for financial support. ; Peer reviewed

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways. ; Tis work was supported by the Government of Canada through the Ontario Research Fund (RE08-067), the Natural Sciences and Engineering Research Council of Canada (NSERC) and by the European Union's Horizon 2020 research and innovation program (773902-SuperPests) awarded to MG and VG; and by the Japan Society for the Promotion of Science KAKENHI (Grant Nos. 18H02203 to TS, 19K22304 to TF and 19K23674 to MT) and the Institute of Global Innovation Research in TUAT to MT, TS, TF and VG. MA was funded through the Global Tesis program, the University of Bari Aldo Moro, Italy.

RNA technologies are the driving forces of modern medicine and biotechnology. They combine the fields of biochemistry, chemistry, molecular biology, cell biology, physics, nanotechnology and bioinformatics. The combination of these topics is set to revolutionize the medicine of tomorrow. After more than 15 years of extensive research in the field of RNA technologies, the first therapeutics are ready to reach the first patients. Thus we are witnessing the birth of a very exciting time in the development of molecular medicine, which will be based on the methods of RNA technologies. This volume is the first of a series. It covers various aspects of RNA interference and microRNAs, although antisense RNA applications, hammerhead ribozyme structure and function as well as non-coding RNAs are also discussed. The authors are internationally highly respected experts in the field of RNA technologies.

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al. ; Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor- SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found. ; This research was supported by grants BFU2011-23896 and the European Union FP7 ANTIGONE project number 278976 (JdlF). NA was funded by Ministerio de Educacion y Ciencia, Spain. VN was funded by the European Social Fund and the Junta de Comunidades de Castilla-La Mancha (Program FSE 2007–2013), Spain. RS was supported by the project Postdok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) and by the Grant 13-12816P (GA CR). OH was supported by the Grant Agency of the Czech Republic grant no. 13-27630P and European Union FP7 MODBIOLIN project number 316304. CR and LBS were supported by the United Kingdom Biotechnology and Biological Sciences Research Council's National Capability Grant to the Pirbright Institute. ; Peer Reviewed

One of the challenges being faced in the twenty-first century is the biological control of plant viral infections. Among the different strategies to combat virus infections, those based on pathogen-derived resistance (PDR) are probably the most powerful approaches to confer virus resistance in plants. The application of the PDR concept not only revealed the existence of a previously unknown sequence-specific RNA-degradation mechanism in plants, but has also helped to design antiviral strategies to engineer viral resistant plants in the last 25 years. In this article, we review the different platforms related to RNA silencing that have been developed during this time to obtain plants resistant to viruses and illustrate examples of current applications of RNA silencing to protect crop plants against viral diseases of agronomic relevance. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation. © 2011. ; The work in the author's laboratory is supported by grants BIO2010-18541 from Spanish MEC and KBBE-204429 from European Union. ; Peer Reviewed

What are the possibilities of DNA and RNA sequences?
Synthetic DNA and RNA sequences are currently being used as nucleic-acid-based drugs for the treatment of a plethora of human diseases under antisense and/or RNA interference therapies. This article from Serge L. Beaucage, Supervisory Research Chemist at the Food and Drug Administration discusses his work with DNA and RNA sequences and the groundbreaking impact this technique could have. Within this Q&A, Beaucage answers questions around the faults with the chemical synthesis process of DNA and RNA sequences, the challenges of purifying DNA and RNA sequences and the technologies you used to help develop this research.

Cationic liposomes are efficient vectors for systemic delivery of therapeutic small interfering RNA (siRNA), taking advantage of RNA interference (RNAi), a naturally occurring gene-silencing mechanism in mammalian cells. However, toxicity at high concentrations, short circulating half-lives and lack of specificity restrict their successful application in a wider scale. The purpose of this study was to evaluate the efficiency of neutral liposomes containing polyethylene glycol (PEG) to encapsulate siRNA in their aqueous core. This formulation will reduce drastically the toxicity associated to cationic liposomes by bringing surface charge to almost zero, increasing stealth degree and therefore circulation time. In this study, we evaluate the efficiency of folate-targeted liposomes for specific delivery of siRNA to activated macrophages, key effector cells in rheumatoid arthritis (RA) pathology which specifically express folate receptor (FR). Myeloid cell leukaemia-1 (Mcl-1) is a protein essential for synovial macrophage survival, since Mcl-1 suppression results in the induction of apoptosis. The effect of MCL1 siRNA incorporated in liposomal formulation was assessed in primary human macrophages and successful inhibition of Mcl-1 expression was achieved. Here we show that the neutral liposomal derived from DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) formulation developed is efficient to encapsulate MCL1 siRNA and silencing gene expression in activated human macrophages. ; Eugénia Nogueira (SFRH/BD/81269/2011) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project "BioHealth − Biotechnology and Bioengineering approaches to improve health quality", Ref. ...