A phosphorothioate oligonucleotide blocks reverse transcription via an antisense mechanism

AbstractWe have studied the inhibition by a phosphorothioate oligodeoxynucleotide (17PScap) of cDNA synthesis performed by either avian or murine reverse transcriptase. Three different mechanisms of inhibition were identified: at low concentrations (⊃100 nM), the cleavage of the RNA template by the retroviral RNase H at the level of the RNA/17PScap duplex accounted for most of the effect, whereas hybrid-arrested cDNA synthesis by an RNase H-independent mechanism marginally contributed to the inhibition. Both mechanisms were sequence-specific. Above 100 nM, the overall cDNA synthesis was reduced in a non-specific manner

Systematic screening of LNA/2′-O-methyl chimeric derivatives of a TAR RNA aptamer

AbstractWe synthesized and evaluated by surface plasmon resonance 64 LNA/2′-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2′-O-methyl nucleoside analogues where one or two LNA units are located on the 3′ side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay

Single-molecule observations of RNA-RNA kissing interactions in a DNA nanostructure

RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures

A Boost for the Emerging Field of RNA Nanotechnology: Report on the First International Conference on RNA Nanotechnology

This Nano Focus article highlights recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland, OH, USA (October 23-25, 2010) (http;//www.eng.uc.edu/nanomedidne/RNA2010/), chaired by Peixuan Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and Its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future funding opportunities at NCl. As indicated by the feedback received from the invited speakers and the meeting participants, this meeting was extremely successful, exciting, and informative, covering many groundbreaking findings, pioneering ideas, and novel discoveries

Aptamers in Bordeaux, 24−25 June 2016

The symposium covered the many different aspects of the selection and the characterization of aptamers as well as their application in analytical, diagnostic and therapeutic areas. Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of “Aptamers in Bordeaux” will take place on September 2017 (http://www.aptamers-in-bordeaux.com/)

NMR structure of a kissing complex formed between the TAR RNA element of HIV-1 and a LNA-modified aptamer

The trans-activating responsive (TAR) RNA element located in the 5′ untranslated region of the HIV-1 genome is a 57-nt imperfect stem-loop essential for the viral replication. TAR regulates transcription by interacting with both viral and cellular proteins. RNA hairpin aptamers specific for TAR were previously identified by in vitro selection [Ducongé,F. and Toulmé,J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605–1614]. These aptamers display a 5′-GUCCCAGA-3′ consensus apical loop, partially complementary to the TAR one, leading to the formation of a TAR–aptamer kissing complex. The conserved GA combination (underlined in the consensus sequence) has been shown to be crucial for the formation of a highly stable complex. To improve the nuclease resistance of the aptamer and to increase its affinity for TAR, locked nucleic acid (LNA) nucleotides were introduced in the aptamer apical loop. LNA are nucleic acids analogues that contain a 2′-O,4′-C methylene linkage and that raise the thermostablity of duplexes. We solved the NMR solution structure of the TAR–LNA-modified aptamer kissing complex. Structural analysis revealed the formation of a non-canonical G•A pair leading to increased stacking at the stem-loop junction. Our data also showed that the introduction of LNA residues provides an enhanced stability while maintaining a normal Watson–Crick base pairing with a loop–loop conformation close to an A-type

Détection du glyphosate par inhibition enzymatique

International audienceLes pesticides sont des substances chimiques largement utilisées en agriculture pour protéger les cultureset les semences avant et après la récolte. Ils ont apporté des bénéfices économiques importants depuisleur mise sur le marché. En même temps, leur généralisation a créé de sérieux problèmes liés à leurseffets sur l’environnement et sur la santé humaine1. En raison de son efficacité à tuer les mauvaisesherbes avec un coût modéré, le glyphosate est l'un des pesticides les plus utilisés au monde. Bien qu’ilsoit considéré moins toxique par rapport aux autres pesticides chimiques, le glyphosate a suscité lapréoccupation publique en raison de son utilisation massive conduisant à sa détection ainsi que cesrésidus dans les eaux de surfaces2. Depuis nos connaissances, aucun système de détection efficace etpeu coûteux de glyphosate n'est actuellement disponible. Un tel système, qui permettrait de détecter lacontamination sur place et en temps réel, est nécessaire de toute urgence pour alerter les autorités localeset sensibiliser la population.Dans cette optique, nous étudions le potentiel de développement d’une méthode rapide, peu coûteuse etécologique pour surveiller la contamination des eaux de surface par le glyphosate. Le principe dedétection est basé sur l'inhibition de l'activité de deux enzymes, l'exonucléase I (Exo I) et la T5exonucléase (T5 Exo) par le glyphosate. Ces deux enzymes digèrent les oligonucléotides en séquencesplus courtes jusqu'aux mononucléotides. En présence du glyphosate dans le milieu réactionnel, l'activitédes deux enzymes est inhibée et la digestion est retardée. En utilisant le SYBR Gold comme sonde defluorescence, la détection du glyphosate est réalisée par spectroscopie de fluorescence : puisque ladigestion enzymatique est freinée, l'intensité de fluorescence de la sonde augmente avec des quantitéscroissantes en glyphosate, ce qui indique la perte de la digestion enzymatique en présence de cecontaminant. Dans les conditions optimales, cette méthode permet la détection spécifique etreproductible du glyphosate dans une gamme de linéarité entre 100 et 500 μM, ce qui ouvre la voie à ladétection éventuelle du glyphosate dans des matrices réelles à des limites de détection plus appropriées