Resonance Raman analysis of a fluorescently labeled oligonucleotide forming a very stable hairpin

Abstract

An oligodeoxynucleotide has been synthesized, which mimics an ``antigene'' oligonucleotide with a polypyrimidic stretch on its 5′ side and is protected on its 3′ side against nucelases by a naturally forming and very stable hairpin, 5′GCGAAGC3′. The in vitro degradation of the resulting oligonucleotide d(5′TTCTCGCGAAGC3′) has already been studied by fluorescence resonance energy transfer (FRET) (Réfrégiers et al. 1996, J Biomol Struct Dyn 14: 365 – 371). The technique required the grafting of fluorophores at both ends of the oligonucleotide. In the present work we have compared the hairpin formed in the presence and in the absence of such fluorophores. This was achieved by the study of the Raman spectra (excitation at 257 nm) of the oligodeoxynucleotides H, which forms the hairpin (5′TTCTCGCGAAGC3′), and a con-trol C (5′TTCTCCGGAAGC3′) which is unable to form the hairpin. Resonance Raman spectroscopy with 257 nm excitation greatly favors the resonance of purines and therefore the study of the 3′ part of the oligonucleotides. The difference spectrum obtained from resonance Raman spectra of C and H showed marker peaks specific for hairpin formation. The search for these marker peaks in difference spectra involving the Raman spectrum of H labeled by fluorophores and either C or H proved that the fluorophores do not modify the structure of the hairpin but only the vibrations of the two terminal bases on which the fluorophores are grafted. The use of such labeling is then justified in order to allow oligonucleotides protected by a hairpin on their 3′ side to be studied by fluorescence spectroscopy.