siRNA and RNAi optimization

The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects (OTEs) such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation, and miRNA-like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. © 2016 Wiley Periodicals, Inc.The work is supported by grants of the European Commission (NMP4-LA-2011-262943, MULTIFUN), the Spanish Ministry of Economy (CTQ2014-52588-R, RTC-2014-2038-1), Generalitat de Catalunya (2014/SGR/624) and the Instituto de Salud Carlos III (CB06_01_0019) with assistance from the European Regional Development Fund.Peer reviewe

RNA/aTNA chimeras: RNAi effects and nucleases resistance of single and double stranded RNAs

The RNA interference pathway (RNAi) is a specific and powerful biological process, triggered by small non-coding RNA molecules and involved in gene expression regulation. In this work, we explored the possibility of increasing the biological stability of these RNA molecules by replacing their natural ribose ring with an acyclic L-threoninol backbone. In particular, this modification has been incorporated at certain positions of the oligonucleotide strands and its effects on the biological properties of the siRNA have been evaluated. In vitro cellular RNAi assays have demonstrated that the L-threoninol backbone is well tolerated by the RNAi machinery in both double and single-stranded fashion, with activities significantly higher than those evinced by the unmodified RNAs and comparable to the well-known phosphorothioate modification. Additionally, this modification conferred extremely strong resistance to serum and 3′/5′-exonucleases. In view of these results, we applied this modification to the knockdown of a therapeutically relevant human gene such as apolipoprotein B ( ApoB). Further studies on the activation of the innate immune system showed that L-threoninol-modified RNAs are slightly less stimulatory than unmodified RNAs.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 Elisa Pedone for her helpful advice and for providing technical assistance.Peer reviewe

RNA modified with acyclic threoninol nucleic acids for RNA interference

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 reviewe

Synthesis and Properties of Oligodeoxynucleotides Carrying 2-Aminopurine

The use of benzoyl, isobutyryl and dimethylaminomethylidene groups for the protection of the exocyclic amino function of 2-aminopurine during oligonucleotide synthesis has been investigated. Best results in the synthesis were obtained with the monomers of 2-aminopurine protected with the isobutyryl groupThis work was supported by the Spanish Ministry of Education (grant CTQ2010-20541) and the Generalitat de Catalunya (2009/SGR/208).Peer reviewe

Solid-phase synthesis of modified oligonucleotides

Synthetic oligonucleotides are ubiquitously found in most laboratories since solid-phase synthesis protocols have become highly optimized. These protocols make it possible to synthesize a large variety of modified oligonucleotides. As one example, we will review some of the developments regarding oligonucleotide synthesis from our own group. In particular, we will describe the synthesis of oligonucleotides carrying non-natural bases, of oligonucleotide-peptide conjugates, and of modified oligonucleotides used in the assembly of nanomaterials.The work summarized in this review has been conducted by a large number of colleagues and collaborators whom I thank for their contributions and friendship. We also thank E.E.C.C., E.M.B.L., the Spanish Ministry of Education and the Fundació La Caixa for funding this work.Peer reviewe

Oligonucleotide delivery: A patent review (2010-2013)

Introduction: The use of aptamers, antisense technology and RNA interference has allowed nucleic acids to be considered as promising alternatives to classical drugs. However, nucleic acids face several obstacles in the creation of effective nucleic acid drugs. The development of these approaches has strengthened the pipeline with an increasing number of these therapies in clinical trials.Areas covered: This review covers research and patent literature from the last three years, focusing on the development of safe and effective non-viral drug delivery systems for the treatment of diseases such as cancer or genetic disorders by using oligonucleotides.Expert opinion: The therapeutic applications of oligonucleotides have overcome multiple obstacles, especially in biodistribution and cellular internalization. Cationic lipids are the most used vehicles for the preparation of novel formulations. Combinatorial libraries of these compounds and the use of solid lipid nanoparticles carrying these synthetic cationic lipids (cholesterol and PEG) have enhanced cellular uptake and biocompatibility of nucleic acids. Besides this extensive use, synthesis of oligonucleotides covalently linked to lipids has also emerged as a promising alternative to formulations. The use of peptides alone or in combination with lipids is an expanding field for oligonucleotide delivery. Polymeric platforms are also good candidates as they showed improved cellular uptake, biodegradability, biocompatibility and the possibility of incorporating several components, such as ligands for receptor-mediated endocytosis and molecules, to facilitate endosomal escape. Finally, nanomaterials may also play an important role in the future. The last developments showed improvement in in vivo efficacy, thus gaining a foothold in therapeutics.This work is supported by the European Commission (Grant NMP4-LA-2011-262943, MULTIFUN), by the Spanish Ministry of Education (Grant CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208) and the Instituto de Salud Carlos III (CB06_01_0019).Peer reviewe

Variable-Temperature Size Exclusion Chromatography for the Study of the Structural Changes in G-Quadruplex

The conformational equilibria of a guanine-rich sequence found at the promoter region of the human c-kit oncogene are studied by means of circular dichroism spectroscopy (CD) and variable-temperature size exclusion chromatography (SEC). It is shown that the wild sequence ckit21 exists as a mixture of monomeric and multimeric G-quadruplexes. Appropriate mutation of several bases in the wild sequence produces the shift from parallel to antiparallel G-quadruplex, as well as the disappearance of multimeric species. The shift from the antiparallel to the parallel conformation induced by temperature is reflected in both CD and SEC profiles.The authors acknowledge funding from the Spanish Government (CTQ2012-38616-C02-02 and CTQ2010-20541-C03-01).Peer reviewe

1-[2,3-Bis(tetradecyloxy)propyl]-3-[2-(piperazin-1-yl)ethyl]urea

Starting from 2,3-bis(tetradecyloxy)propan-1-amine (1), the synthesis of the target compound 1-[2,3-bis(tetradecyloxy)propyl]-3-[2-(piperazin-1-yl)ethyl]urea (2) is reported. The title compound was characterized by 1H-NMR, 13C-NMR and ESI/MS analysis.This work is supported by grants of the Spanish Ministry of Economy (MINECO) (CTQ2014-52588-R, RTC-2014-2038-1, CTQ2014-61758-EXP), Generalitat de Catalunya 2014/SGR/624) and the Instituto de Salud Carlos III (CB06_01_0019). 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 acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Spectroscopic study of the interaction of actinomycin D with oligonucleotides carrying the central base sequences -XGCY- and -XGGCCY- using multivariate methods

10 pages, 5 figures, 1 table.-- PMID: 17123067 [PubMed].-- Published online Nov 23, 2006.Supporting information (4 pages, 3 suppl. figures) available at: http://www.springerlink.com/content/870650451902431q/MediaObjects/216_2006_946_MOESM1_ESM.docThe interactions of actinomycin D (ACTD) with the oligonucleotides 5′-CAAAGCTTTG-3′, 5′-CATGGC CATG-3′ and 5′-TATGGCCATA-3′ were investigated by means of acid–base titrations and mole-ratio and melting experiments monitored by molecular absorption and circular dichroism (CD) spectroscopies. For each experiment, CD and molecular absorption spectra were recorded at each point in the experiment, and later analyzed via appropriate multivariate data analysis methods. The study of the interactions between these oligonucleotides and ACTD at 25°C showed the formation of an interaction complex with a stoichiometry of 1:1 (ACTD:duplex) and values for the log(formation constant) of 5.1 ± 0.3, 6.4 ± 0.2, and 5.6 ± 0.2, respectively. An additional interaction complex at higher temperatures was also detected, which might be related to the single-stranded forms of the oligonucleotides.We acknowledge two grants from the Spanish Ministerio de Educación y Ciencia (projects BFU2004-02048/BMC and BQU2003-0191).Peer reviewe

Synthesis of labelled PNA oligomers by a post-synthetic modification approach

The preparation of t-butoxycarbonyl (Boc)-protected O4-(o-nitrophenyl) thymine peptide nucleic acid (PNA) monomer is described. This PNA monomer was incorporated into PNA oligomer sequences. The post-synthetic modification of the oligomers to yield fluorescently-labelled PNA oligomers was studied before and after the removal of the protecting groups. In both cases, the desired fluorescently-labelled PNA oligomer was obtained in good yields.This work was supported by the Commission of the European Union as part of the Information Societies Technology Programme (IST-1999-11974) by the Dirección General de Investigación Científica y Técnica (grants BQU2000-0649) and the Generalitat de Catalunya (2000-SGR-0018 and 2001-SGR-0049).Peer reviewe