Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Improved binding of pyrimidine a-DNAs with phosphoramidate linkages to their DNA targets studied by FTIR and UV spectroscopy.

Oligonucleotides provide the best specificity of recognition of nucleic acid sequences known to date. Numerous modifications, including base and backbone modifications, have been proposed to increase the affinity of artificial oligonucleotides to their targets. In particular the replacement of the phosphodiester linkages of the b-deoxynucleotides by uncharged linkages has been used hoping that a decrease of the electrostatic repulsion between the different negative partners could favor their binding. However b-oligomers with methylphosphonate or N-alkyl phosphodiester amidate linkages do not show higher affinity for single or double stranded nucleic acids than normal oligonucleotides.

More recently, it has been proposed that the expected stabilization could be obtained if in addition to backbone modification, the anomeric configuration of the nucleosides is changed from b to a. We have studied by UV thermal denaturation.and FTIR spectroscopy the binding of pyrimidine N-alkylphosphoramidate a-oligonucleotides to their (b-ODNs) targets. (aT15M) and (aT15MÕ) bind the bA15 strand through reverse Watson-Crick hydrogen bonds to form a very stable antiparallel duplex (DTm +18 or 14¡C for M = N-[2-methoxyethyl] phosphoramidate or MÕ = N-[2-propyl] phosphoramidate linkages respectively as compared to bb analogues). The relative orientation of the duplex strands having an (aT15M), (aT15MÕ) or (aT15) strand is antiparallel instead of parallel with mixed sequence pyrimidine a-ODNs.

While (bT15) failed to form a triplex with a long (bdAn.bdTn) duplex, the Tm of the Hoogsteen part of the triplex formed by (aT15M) or (aT15MÕ) reaches 40 and 30¡C respectively. Not only phosphoramidate a-deoxythymidylates but phosphoramidate a-ODNs containing cytosines and thymines form much more stable triplexes than phosphodiester b-ODNs did, even at pH 7 (DTm +20¡C for N-[2-methoxyethyl]-phosphoramidate, DTm +10¡C for phosphoromorpholidate pyrimidine a-ODNs, 12mer (aCTM) and (aCTMo) at pH7). All (aCTM), (aCTMo), (aT15M), (aT15MÕ) third strands are found antiparallel in contrast to (aT15) which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides combining both replacements might contribute to the improve stability of the triplexes. This stability with a great degree of resistance against nuclease degradation make these modifications attractive for development of oligonucleotides for gene regulation.

Bei-Wen Sun à, Fr?d?ric Geinguenaud à, Eliane Taillandier à* Magali Naval ¤, Alain Laurent ¤, Fran?oise Debart ¤, Jean-Jacques Vasseur ¤

àLaboratoire de Spectroscopie Biomol?culaire, Universit? Paris Nord, France. Tel : 01 48 38 76 90 ; Fax : 01 48 37 74 43 ; email : eliane.taillandier@smbh.univ-paris13.fr
¤Laboratoire de Chimie Organique Biomol?culaire de Synth?se, Universit? Montpellier II, France Tel : 04 67 14 38 98 ; Fax : 04 67 04 20 29 ; email : vasseur@univ-montp2.fr