RESONANCE RAMAN ANALYSIS OF A FLUORESCENTLY LABELED OLIGONUCLEOTIDE FORMING A VERY STABLE HAIRPIN

An oligodeoxynucleotide has been synthesized, which mimics an ''antige ne'' oligonucleotide with a polypyrimidic stretch on its 5' side and i s protected on its 3' side against nucelases by a naturally forming an d very stable hairpin, 5'GCGAAGC3'. The in vitro degradation of the re sulting oligonucleotide d(5'TTCTCGCGAAGC3') has already been studied b y fluorescence resonance energy transfer (FRET) (Refregiers et al. 199 6, J Biomol Struct Dyn 14: 365-371). The technique required the grafti ng of fluorophores at both ends of the oligonucleotide. In the present work we have compared the hairpin formed in the presence and in the a bsence of such fluorophores. This was achieved by the study of the Ram an spectra (excitation at 257 nm) of the oligodeoxynucleotides H, whic h forms the hairpin (5'TTCTCGCGAAGC3'), and a control C (5'TTCTCCGCAAG C3') which is unable to form the hairpin. Resonance Raman spectroscopy with 257 nm excitation greatly favors the resonance of purines and th erefore the study of the 3' part of the oligonucleotides. The differen ce spectrum obtained from resonance Raman spectra of C and H showed ma rker 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 tw o terminal bases on which the fluorophores are grafted. The use of suc h labeling is then justified in order to allow oligonucleotides protec ted by a hairpin on their 3' side to be studied by fluorescence spectr oscopy.