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     High throughput synthesis of long oligonucleotides (Platform)

  Director : Gouyette Catherine (oligos@pasteur.fr)



In our platform, we make exclusively oligonucleotides synthesis but different types of synthesis :

- in 96-well plate format, for DNA microarrays, and in this case oligonucleotides are about 70 bases long (purified quickly on C18 cartridges, checked by analytical HPLC and capillar electrophoresis)

- synthesis made one by one of oligonucleotides of different sizes, modified or not, purified or not by HPLC, and in amounts going from a few DO units (at 260nm) to some milligrams

- synthesis of purified RNA or siRNA



a) First of all, the sequences we made in 96-well plates, for programs agreed by the genopole steering commitee :

- We end up 800 oligos that were left for Plasmodium falciparum, started in 2003, and also the last oligos (begun in 2003) for virulence analysis of the human parasite Entamoeba histolytica with help of DNA microarrays.

-To study the transcriptome of the pathogenic yeast Candida glabrata, we had to synthesize 5910 oligos (70mers) for this project. All the sequences were splitted in lots of 384-well plates and sent in different laboratories in the world (UK,USA, Ireland, Japan) part of the Candida consortium laboratories.

- Gene expression outline of Bacillus anthracis in culture conditions as if in host infection : we only made about 500 oligos for this project which was started before the platform activity .

- Functionnal genomic of Legionella pneumophila to study interactions between the host and the pathogen : about 4200 oligos , some were specific of two particular strains (Columbia and Lens)

b) As, in the past we were making HPLC purified oligos in the Organic Chemistry Unit, we continue this activity for specific needs of laboratories whom we always worked with as:

- Dr Oleg Melnyk in Pasteur Institute in Lille on oligonucleotides functionnalized with an oxoaldehyde group in 5' or 3'and their fixing in a covalent way on DNA microarrays. For this project we make also aminoC6, Cy3 and Cy5 substituted oligos in 5'position.

- Professor Subirana in University of Barcelona (spain) to whom we made a lot of short oligos (from 6 to 14 bases) for cristallography and Xray studies. Generally we make a few milligrams of each sequence, HPLC purified and MALDI-TOF controlled.

- In the scope of a new collaboration with Dr Frédéric Ariey, Pasteur Institute in Cambodgia, to whom we made some Cy3 and Cy5 oligos in view of making Diagnostic DNA microarrays to identify different Plasmodium strain.

c) We work also with many laboratories of the Institute, making RNA and many more siRNA( short interfering RNA) sequences.

The RNA interference (RNAi) phenomenon is a recently observed process in which small double-stranded RNA molecules induce sequence-specific degradation of homologous single strand mRNA.

For this purpose we got some money from the scientific coordination of the business development and industrial partnership department with the enterotrop viruses and antiviral strategy laboratory (Direction F.Golbère-Garapin, Virology Department) to synthesize siRNAs on the following subject :extinction by siRNA of the multiplication of RNA positive viruses of medical interest (enteroviruses, hepatitis C virus).

d) Now, we are starting new projects with two laboratories : one in Pasteur Institute (in the Nuclear Cell Biology unit), the other one in the great East Canceropole (in the biological ressources center , CHRU, Nancy).

In those subjects, w'll have to make " molecular beacons ", which are sequences with a fluorophore at one end (generally 5') and a quencher at the other end of the sequence (3'). When the oligo is alone, it stays as an " hairpin " conformation and there is no fluorescence emitted. When the oligo is hybridized with its target, the extremities are separated and so, there is fluorescence emitted.

Keywords: oligonucleotides, siRNA, modified DNA sequences, molecular beacon

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      Huteau Valerie Superior Technician vhuteau@pasteur.fr

Helynck Olivier Superior Technician ohelynck@pasteur.fr

Activity Reports 2004 - Institut Pasteur

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