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

Synthesis and Triple-Helix-Stabilization Properties of Branched Oligonucleotides Carrying 8-Aminoadenine Moieties

The synthesis of several branched oligonucleotides, i.e., of the parallel hairpins 5-8 and the Y-shaped 9 is described, together with their use in the formation of pyrimidine purine triple helices. Special attention was paid to the optimization of the assembly of the second strand from asymmetric branching molecules. The presence of 8-aminoadenine moieties in the Watson-Crick purine strand and 2′-O-methyl-RNA in the Hoogsteen pyrimidine strand produced strong stabilization of the triplex.Peer reviewe

Fundamental aspects of the nucleic acid i-motif structures

The i-motif structure is formed in cytosine-rich sequences, its building block being the cytosine·cytosine + base pair. This structure is particularly stable at pH values below physiological (∼7.4) and, because of that, it has not attracted as much biological interest as other non-canonical structures such as the G-quadruplex. Nowadays, the proposal of potential roles in vivo, as well as nanotechnological applications, has produced an increasing interest in its study. In this context, the present work provides an overall picture of the i-motif structure. Those aspects related to formation and stability, such as chemical modifications or the interaction with ligands, are discussed. Special attention has been made to the i-motif structures that could have a hypothetical role in vivo, such as those present near the promoter region of several oncogenes.We acknowledge funding from the Spanish government (CTQ2012-38616-C02-02 and CTQ-2010-20541-C03). Sanae Benabou thanks the Spanish Ministerio de Economía y Competitividad for a PhD grant.Peer reviewe

Synthesis and G-Quadruplex-Binding Properties of Defined Acridine Oligomers

The synthesis of oligomers containing two or three acridine units linked through 2-aminoethylglycine using solid-phase methodology is described. Subsequent studies on cell viability showed that these compounds are not cytotoxic. Binding to several DNA structures was studied by competitive dialysis, which showed a clear affinity for DNA sequences that form G-quadruplexes and parallel triplexes. The fluorescence spectra of acridine oligomers were affected strongly upon binding to DNA. These spectral changes were used to calculate the binding constants (K). Log K were found to be in the order of 4–6

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background: G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging. Methods: Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA-drug interactions. Results: We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines. Conclusions: Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed. General significance: The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties. © 2012 Elsevier B.V.This research was supported by the Spanish Ministerio de Ciencia e Innovación (grant numbers CTQ2009-11572 and CTQ2010-20541-C03-01), and the Generalitat de Catalunya (grant numbers 2009-SGR-45 and 2009- SGR-208).Peer reviewe

Stepwise synthesis of oligonucleotide-peptide conjugates containing guanidinium and lipophilic groups in their 3'-termini

Two different series of oligonucleotide-peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide-phosphorothioate conjugates were evaluated as potential antisense oligonucleotides in the inhibition of the luciferase gene

Trimethylguanosine nucleoside inhibits cross-linking between snurportin 1 and m3G-capped U1 snRNA

Macromolecular nuclear import is an energy-and signal-dependent process. The best characterized type of nuclear import consists of proteins carrying the classical NLS that is mediated by the heterodimeric receptor importin α/β. Spliceosomal snRNPs U1, U2, U4, and U5 nuclear import depend both on the 5' terminal m3G (trimethylguanosine) cap structure of the U snRNA and the Sm core domain. Snurportin 1 recognizes the m3G-cap structure of m3G-capped U snRNPs. In this report, we show how a synthesized trimethylguanosine nucleoside affects the binding of Snurportin 1 to m3G-capped U1 snRNA in a UV-cross-linking assay. The data indicated that TMG nucleoside is an essential component required in the recognition by Snurportin 1, thus suggesting that interaction of Snurportin 1 with U1 snRNA is not strictly dependent on the presence of the whole cap structure, but rather on the presence of the TMG nucleoside structure. These results indicate that the free nucleoside TMG could be a candidate to be an inhibitor of the interaction between Snurportin 1 and U snRNAs. We also show the behavior of free TMG nucleoside in in vitro U snRNPs nuclear import. Copyright © Taylor & Francis Group, LLC.This work was supported by Plan Nacional BFU2005-00701 and the Polish Committee for Scientific Research (KBN) # 6 P04A 055 17. D.B. was a recipient of a CNPq Brazilian fellowship and EMBO and FEBS short-term fellowshipsPeer Reviewe

Stepwise synthesis of RNA conjugates carrying peptide sequences for RNA interference studies

Oligoribonucleotide conjugates carrying nuclear localization peptide sequences at the 3?-end were prepared stepwise on a single support. The siRNA duplex carrying the nuclear localization peptide sequence at the 3?-end of the passenger strand has similar inhibitory properties as those of unmodified or cholesterol-modified RNA duplexes.Oligoribonucleotide conjugates carrying nuclear localization peptide sequences at the 3?-end were prepared stepwise on a single support. The siRNA duplex carrying the nuclear localization peptide sequence at the 3?-end of the passenger strand has similar inhibitory properties as those of unmodified or cholesterol-modified RNA duplexes.Peer reviewe

A comparative study of supports for the synthesis of oligonucleotides without using ammonia

A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2(2-nitrophenyl)ethyl, 9-fluorenylmethyl, and 2-nitrobenzyl supports in 0.5M DBU solutions is described. A decrease in cleavage efficiency is observed when small oligonucleotides containing thymidine are linked to the supports. In these conditions oxalyl supports gave the best yields followed by 2-(2-nitrophenyl)ethyl and 9-fluorenylmethyl supports.We are grateful to CICYT (PB92-0043) and E.E.C.C. Biomedicine and Health Programme (BMH1-CT93-1669) for financial support. We thank Drs. Matthias Mann, Gitte Neubauer, Matthias Wilm (EMBL) and Irene Fernández (University of Barcelona) for obtaining mass spectra.Peer reviewe

Synthesis of oligonucleotides carrying anchoring groups and their use in the preparation of oligonucleotide-gold conjugates

Oligodeoxynucleotide conjugates 1-15 carrying anchoring groups such as amino, thiol, pyrrole, and carboxy groups were prepared. A post-synthetic modification protocol was developed. In this method 2′-deoxy-O4-(p-nitrophenyl)uridine-3-phosphoramidite was prepared and incorporated in oligonucleotides. After assembly, the modified nucleoside was made to react with different amines carrying the anchoring groups. At the same time, protecting groups were removed to yield the desired oligonucleotide conjugates. In a second approach, amino, thiol, and carboxylic groups were introduced into the 3′-end of the oligonucleotides by preparing solid supports loaded with the appropriate amino acids. Oligonucleotide-gold conjugates were prepared and their binding properties were examined.Peer reviewe