Effect of RNA secondary structure and modified bases on the inhibition of trypanosomatid protein synthesis in cell free extracts by antisense oligodeoxynucleotides.

Every messenger RNA from leishmanias and trypanosomes has at its 5' end a conserved region termed the mini-exon sequence which, however, varies from species to species. In a systematic study mRNAs from Trypanosoma brucei, Trypanosoma vivax, and Leishmania enriettii were translated in cell-free extracts in the presence of oligodeoxynucleotides complementary to part of the mini-exon sequence. The affinity of the same oligonucleotides for target and non-target mRNAs was determined by thermal elution of filter-bound complexes showing that the critical temperature of half-dissociation of the complexes was linearly related to log (l + x), where l is the length of the oligomer and x its G + C content. A few oligomers exhibited a lower Tc value than expected which was ascribed to the presence of modified RNA bases or to the existence of a hairpin structure in the L. enriettii mini-exon. In most cases the efficiency of translation inhibition by the oligonucleotides was clearly correlated to their affinity for the target RNA. The modified bases weakened the inhibition of protein synthesis by oligonucleotides complementary to these regions

Les oligonucléotides antisens : outils de génétique moléculaire et agents thérapeutiques

La fixation d’un oligonucléotide synthétique, appelé antisens, sur la séquence complémentaire d’un ARN messager choisi pour cible peut conduire à l’inhibition de la synthèse de la protéine correspondante. Il s’agit donc là d’un moyen de réguler artificiellement et de façon spécifique l’expression du gène cible. Deux mécanismes rendent compte de cet effet : d’une part un blocage physique de la progression du ribosome prévient l’initiation de la traduction, d’autre part la dégradation de l’ARNm associé à l’oligonucléotide antisens par les RNase-H empêche l’élongation de la chaîne peptidique. Des modifications chimiques peuvent être introduites dans ces molécules afin de leur conférer des propriétés particulières (résistance aux DNases, haute affinité, pénétration facilitée dans les cellules vivantes). Les oligonucléotides antisens, outils de génétique moléculaire, ont aidé à la caractérisation de la séquence mini-exon, acquise par trans-épissage, présente à l’extrémité 5' de tous les ARNm de trypanosomatidés et à celle de quelques ARNm de nématodes. De plus, des oligonucléotides antisens modifiés, complémentaires de cette séquence mini-exon induisent la mort de Trypanosoma brucei en culture; ces molécules pourraient donc constituer des prototypes d’une nouvelle classe d’agents thérapeutiques

More at stake in stem-cell patents

Austin Smith and others argue in favour of patenting technologies derived from human embryonic stem cells (Nature 472, 418; 2011), a case still pending with the European Court of Justice. But there is more at stake than European commercial interests. In 1998, agreement was reached in Europe under Directive 98/44 not to recognize patents involving the use of human embryos for commercial purposes (R. Hipp and P. Liese Nature 474, 36; 2011). The court must decide whether the use of these cells \u2018necessitates the prior destruction of human embryos or their use as base material\u2019, as the advocategeneral, Yves Bot, has argued (see go.nature.com/gsap8n). If so, such use would seem to fall beyond the scope of what is legally patentable. Smith et al. warn that \u201cEuropean discoveries could be translated into applications elsewhere, at a potential cost to the European citizen.\u201d This begs the question of whether patents, which may also be held by non-European companies, may sometimes impede wider research cooperation (S. Rabin Nature Biotechnol. 23, 817\u2013819; 2005). In any case there will often be some commercial risk whenever Europe defends a more rigorous ethical standard than is defended elsewhere. This risk is not itself an argument against upholding the standard prescribed by law. Without prejudice to the final judgment in this case, the resolution of patent law is and ought to be more than a question of European commercial interest