Michel Thibault, Debart Françoise, Vasseur Jean-Jacques, Geinguenaud Frédéric, Taillandier Elaine
Laboratoire de Chimie Organique Biomoléculaire de Synthese, UMR 5625 CNRS-UMII, CC 008, Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex, France.
J Biomol Struct Dyn. 2003 Dec;21(3):435-45. doi: 10.1080/07391102.2003.10506938.
The triplexes formed by pyrimidine alpha-oligodeoxynucleotides, 15mers alpha dT(15) or 12mers alpha dCT 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 beta-oligodeoxynucleotides (beta-ODNs). Among the alpha-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (alpha dCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the alpha dCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All alpha dCTPN 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 degrees C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65 degrees C). While unfavorable steric effects are overcome with alpha anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate alpha-thymidilate strands (alpha dT(15)PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to beta dA(15) either of alpha dT(15)PN as duplex strand and beta dT(15) as third strand, or of two hydrophobic alpha dT(15)PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the alpha dT(15)PN binds as Hoogsteen third strand.
通过紫外光谱和傅里叶变换红外光谱研究了由嘧啶α-寡脱氧核苷酸形成的三链体，即具有与DNA双链靶标相连的二甲氧基乙基（PNHdiME）、吗啉代（PMOR）或丙基（PNHPr）非离子型磷酰胺酯键的15聚体α-dT(15)或12聚体α-dCT。由于相同长度的互补链之间静电排斥力的降低，所有修饰导致形成的三链体比用未修饰的磷酸二酯β-寡脱氧核苷酸（β-ODN）形成的三链体更稳定。在具有C和T碱基以及非离子型磷酰胺酯键（α-dCTPN）的α-ODN第三条链中，最有效的修饰是（PNHdiME）。以非对映异构体混合物形式获得的α-dCTPN第三条链稳定性增强，但会因PMOR键的空间位阻或PNHPr键的疏水性而减弱。所有α-dCTPN链即使在中性pH下也能形成三链体。在最有利的情况（PNHdiME）下，我们通过傅里叶变换红外光谱表明，在pH 7时形成的三链体由Hoogsteen T*A.T三联体以及第三条链的G的O6与C之间的氢键维持（Tm = 30℃）。在pH 6时检测到质子化胞嘧啶与三链体的高度稳定化相关（Tm = 65℃）。虽然α-异头物克服了不利的空间效应，但pH依赖性的限制并未完全消除。对于与相同长度的靶标双链相互作用的非pH依赖性磷酰胺酯α-胸苷酸链（α-dT(15)PN），证明了不同的三链体。在低离子强度和DNA浓度下，我们观察到α-dT(15)PN作为双链链与β-dA(15)结合，β-dT(15)作为第三条链，或者观察到两条疏水的α-dT(15)PNHPr链结合。DNA和抗衡离子浓度的增加会使阴离子靶标双链稳定，然后α-dT(15)PN作为Hoogsteen第三条链结合。
