Valerie Helin

Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.

Uptake and intracellular distribution of oligonucleotides vectorized by a PAMAM dendrimer.

We studied the uptake and intracellular distribution of an FITC labelled phosphodiester oligodeoxynucleotide (ODN) vectorized by a dendrimeric structure in cell culture.

Real-time detection and efficacy of antisense oligonucleotides delivered by PAMAM dendrimers in living cells

The aim of the present investigation was to study the behavior of PAMAM dendrimer-nucleic acid complexes in vitro and living cells. We demonstrated the rapid and sensitive detection of mRNA in living cells using molecular beacon pair, one with a donor and the other with a quenching fluorophore that hybridises to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). The molecular beacon was composed of a 13-nt loop structure containing the antisense sequence that can hybridise with the AUG translational start site of the Friend env gene. It was shown that SuperFect may stimulate the antisense ON-RNA hybridisation. The secondary structure of antisense oligonucleotide was optimized. An antisense sequence-specific inhibition of 75% was obtained for one reporter gene with a stem-loop ODN containing four phosphorothioate groups, two at each end.

Development of a Rapid Screening System to Test Antisense ODN Modifications and Carriers

Abstract We developed a rapid screening system, using two reporter genes under the control of the same promoter, to identify the biological activity of modified or/and vectorized antisense oligodeoxynucleotides (ODNs). The ability of a dendrimeric structure and a monocationic cholesterol derivative to enhance ODN cellular uptake was previously investigated by fluorescence analysis. Then, the assay system was validated through investigating the effect of both vectors on antisense ODN efficiency.