Conversation 11: No. 2

category image Volume: Conversation 11
Issue Number 2
May 2000
ISBN 0-940030-81-0

Targeting of Pu.Py Duplexes by GA and GT Rich Oligonucleotides on Microchip and in Solution

Formation of triple helices with GA and GT third strands has been studied. Besides the usual investigation techniques common for characterizing triple helical formation (CD spectroscopy, gel shift mobility assay, chemical probing and S1 nuclease footprinting) we have used a new technique in which targeting of polypurine sequences in duplexes was demonstrated on oligonucleotide microchips. This technique is very successful to quickly test a large number of potential triple helix formation. In this work we used oligonucleotide microarray to study the specificity of DNA duplex recognition by GA and GT strands. Generic 6-mer microchip containing all possible 46 = 4,096 single-stranded hexadeoxyribonucleotides immobilized within individual gel pads was applied. To study formation of intermolecular triple helices on the generic microchip, a number of Pu.Py duplexes were formed by hybridization of the mixture of purine octadeoxyribonucleotides on the microchip followed by targeting of the duplexes by GA or GT third strands. Melting behavior of the formed structures was investigated using fluorescence measurements under microscope. In solution we present the results obtained for GT triplexes and discuss the characteristics of the CD spectra. Results obtained by S1 nuclease footprinting, KMnO4 and DMS chemical probing are consistent with the spectroscopic data reflecting triplex structure formation.

E. Khomyakova1,2,3
J. Liquier2
T. Huynh-Dinh4
V. Florentiev1
A. Mirzabekov1,3
and E. Taillandier2

1 Engelhardt Institute of Molecular Biology
Russian Academy of Sciences
32 Vavilov St.
117984 Moscow, Russia
2Laboratoire de Spectrocopie Biomoleculaire
Universite Paris Nord
74 rue Marcel Cachin
F93017 Bobigny Cedex, France
3Argonne National Laboratory
Argonne, IL 60439 USA
4Unite de Chimie Organique
Institut Pasteur
28 rue du Docteur Roux
75724 Paris Cedex 15, France


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