«SMALL RNAs and APTAMERS»        Jean-Jacques Toulmé

                                                                                                            Group Leader

 

Over the last thirty years several strategies have been worked out in our group that make use of oligonucleotides for artificially regulating gene expression: antisense oligomers, ribozymes and more recently aptamers. J.-J. Toulmé team contributed to the development of the antisense strategy, in particular to the roled played by RNaseH in the oligodeoxynucleotide-mediated effects. The in vitro development of protozoan parasites -trypanosomes and leishmanias- was selectively prevented by chemically-modified oligomers complementary to the mini-exon sequence located at the very 5’ end of every protozoan mRNA.



FroM antisense oligonucleotides...

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RNA structures contribute to the regulation of gene expression. Structured motifs might either behave as regulatory signals per se or constitute  binding sites of regulatory protein. Numerous RNA hairpins are involved in the control of gene expression in pathogenic viruses. The trans-activating responsive (TAR) element of the human immunodeficiency virus (HIV) or the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) are examples of such regulatory RNA elements.




... to aptamers.

Cultured murine macrophages infected by L. amazonenzis, treated by antisense oligonucleotide

Ligands that would specifically bind to an RNA hairpin with high affinity might interfere with the function of the hairpin. In the case of regulatory viral RNA hairpins such ligands might constitute anti-viral agents.




The TAR RNA hairpin of HIV-1 is

recognized by cellular and viral proteins.

Recognition of an RNA sequence with an oligonucleotide is easily achieved through Watson-Crick base-pairing. However antisense-based strategies are poorly adapted to the targeting of RNA structures. Intramolecular interactions engaged in RNA folding compete with the intermolecular association of the RNA target with its complementary sequence, thus weakening or even preventing the formation of the antisense-sense double-stranded complex.

Instead, we made use of in vitro selection for the identification of oligonucleotides able to interact with folded RNA hairpins. SELEX (Systematic evolution of ligands by exponential enrichment) was carried out to this aim against RNA hairpins playing a pivotal role in HIV or HCV lifecycle. Aptamers were identified for both the trans-activating responsive (TAR) hairpin of HIV-1 and stem-loop structures of the 5' or 3' untranslated regions of the HCV mRNA

Anti-TAR DNA and RNA aptamers were identified and fully characterized. These aptamers are hairpins that display a sequence in the apical loop partially complementary to the TAR one. The resulting loop-loop interaction, so-called kissing complex, generates an intermolecular helix stacked between the stem of each hairpin. Both RNA and chemicaly-modified anti-TAR aptamers inhbit in vitro TAR dependent  gene expression.

Recognizing RNA hairpins

biotechnological developments of aptamers

In situ specific inhibition of TAR-dependent  beta galactosidase expression in  HeLa cells by RNA aptamer.

The binding properties of aptamers (exquisite specificity and strong affinity) make them rival of antibodies. They can be quite easily converted into biotechnoogical tools adapted to many different purposes. Indeed we develop aptamer-based probes and analytical devices for diagnostic purposes. More applications are carried out by the associated technology unit Novaptech (see technology transfer page).