UV SPECTROSCOPY OF DNA IN TRIPLE HELIX

C. M. Muntean1, J. Liquier2 and E. Taillandier2

1National Institute of Research and Development for Isotopic and Molecular Technologies,  R-400293 Cluj-Napoca, P.O. 5, Box 700, Romania
2Laboratoire de Spectroscopie Biomolculaire,  CNRS  URA  1430, UFR  Sant Mdecine Biologie Humaine, Universit Paris-Nord, 74, rue Marcel Cachin,  93017 Bobigny, France   

		Over the past few years, oligonucleotides have received increased attention as a potential new class of pharmacologically active compounds [1]. They can be used to interfere with gene expression in a sequence-specific manner by targeting either mRNA via double helix formation or chromosomal DNA via triple helix formation.
		The so-called antigene strategy is based on local triple helix formation via the binding of an oligonucleotide to the major groove of double-helical DNA [1]. 
		This work is a study of UV spectroscopy, concerning the formation and stabilization of DNA in triple helix, in the presence of Zn2+ and Na+ ions. The third strand of this triple helix contains guanines and thymines and has a parallel orientation, with respect to the polypurinic strand of the Watson-Crick double helix.
		In this study we have used the triple helix forming oligonucleotide by twice folding 5'-GGTGGGTGGTG - linker - CTCCTCCCTCCT - linker - AGGAGGGAGGAG-3', where  the linker = pO(CH2CH2O)3p. In the formed triple helix, triplets are characterized by a purine / pyrimidine motif. 
		Double and triple helix stabilities were measured by thermal DNA melting experiments. They were carried out on an Uvikon 933 spectrophotometer, using quarz cuvettes of 1 cm optical pathlength. UV absorbance spectra and absorbance versus temperature profiles were measured at 258, 280 and 330 nm. All  melting curves of the samples were obtained upon heating or cooling. The temperature of the cell holder was varied by circulating liquid using a Huber waterbath controlled by a Huber programmer. Temperature monitoring was achieved by using a thermocouple in a control cuvette. 
		The melting temperature (Tm) was evaluated as the temperature of half-association (or half-dissociation) of the formed complexes determined by the first derivative of the melting curve [1]. In the case of triplexes, a biphasic profile has been found, with a first transition from triplex to duplex and a second transition from duplex to open strand.    

References

1.	Zhou-Sun, B.-w, Sun, J.-s, Gryaznov, S. M., Liquier, J., Garestier, T., Hélène, C., and Taillandier, E., A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3'- P5' phosphoramidate linkages, Nucleic Acids Research, 25: 1782-1787 (1997).