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

Abstract

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 microairay 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.