FTIR and UV Spectroscopy Studies of Triplex Formation Between α-Oligonucleotides with Non-Ionic Phoshoramidate Linkages and DNA Targets

Abstract

The triplexes formed by pyrimidine α-oligodeoxynucleotides, 15mers αdT₁₅ or 12mers adCT having dimethoxyethyl (PNHdiME), morpholino (PMOR) or propyl (PNHPr) non- ionic phosphoramidate linkages with DNA duplex targets have been investigated by UV and FTIR spectroscopy. Due to the decrease in the electrostatic repulsion between partner strands of identical lengths all modifications result in triplexes more stable than those formed with unmodified phosphodiester β-oligodeoxynucleotides (β-ODNs). Among the α-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (αdCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the αdCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All αdCTPN strands form triplexes even at neutral pH. In the most favorable case (PNHdiME), we show by FTIR spectroscopy that the triplex formed at pH 7 is held by Hoogsteen T*A.T triplets and in addition by an hydrogen bond between O6 of G and C of the third strand (Tm = 30° C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65° C). While unfavorable steric effects are overcome with a anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate a- thymidilate strands (adT15PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to βdA15 either of αdT15PN as duplex strand and βdT15 as third strand, or of two hydrophobic αdT15PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the αdT15PN binds as Hoogsteen third strand.