Aboul-ela F, Murchie A I, Homans S W, Lilley D M
Department of Biochemistry, University, Dundee, U.K.
J Mol Biol. 1993 Jan 5;229(1):173-88. doi: 10.1006/jmbi.1993.1016.
The three-dimensional structures of a DNA oligonucleotide containing three extra unpaired adenosine residues (dGCCAGGAAATGGAC+dGTCCGACCTGGC) and that of the perfect duplex analogue (dGCCAGGTCGGAC+dGTCCGACCTGGC) have been studied in solution by 1H and 13C nuclear magnetic resonance. All non-exchangeable aromatic and H-1', H-2', H-2" sugar protons were assigned using standard assignment pathways for B-DNA. All cross-peaks within these pathways were present for the perfect duplex molecule as would be expected for a right-handed A or B-form duplex. However, a few cross-peaks which would be expected in the standard case are extremely weak in the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of the bulged duplex even at long mixing times (250 ms). For example, almost no cross-relaxation is observed between the H-6 proton of C22 and the H-1' of A21, directly across from the three base bulge. Yet the continuity of assignment pathways through the three base bulge argues against any discontinuous "looping out" of one or more of the extra adenosine residues. Double quantum-filtered correlated spectroscopy experiments demonstrate very little deviation from south sugar conformations for residues at or near the bulge. The perfect duplex contains three A.T basepairs as expected, resulting in three very intense T imino-AH2 cross-peaks in the H2O NOESY experiment. In contrast, only two such intense cross-peaks are observed in the same experiment using the bulged duplex sample. Assignments of the two T imino peaks using one-dimensional NOEs are consistent with disruption of the T.A base-pair immediately 3' to the bulge; this is consistent with our earlier observation of chemical reactivity at a T 3' to an An or Tn bulge. We also find evidence of disruption of the G.C base-pair immediately 5' to the bulge.
通过氢-1和碳-13核磁共振在溶液中研究了含有三个额外未配对腺苷残基的DNA寡核苷酸（dGCCAGGAAATGGAC + dGTCCGACCTGGC）及其完美双链类似物（dGCCAGGTCGGAC + dGTCCGACCTGGC）的三维结构。使用B-DNA的标准归属途径对所有不可交换的芳香族质子以及H-1'、H-2'、H-2''糖质子进行了归属。对于完美双链分子，这些途径内的所有交叉峰均存在，这与右手A或B型双链的预期情况相符。然而，即使在长混合时间（250毫秒）下，凸起双链的核Overhauser增强光谱（NOESY）谱中一些在标准情况下预期会出现的交叉峰也极其微弱。例如，在三个碱基凸起正对面，几乎未观察到C22的H-6质子与A21的H-1'之间的交叉弛豫。然而，通过三个碱基凸起的归属途径的连续性表明，不存在一个或多个额外腺苷残基的任何不连续“环出”情况。双量子滤波相关光谱实验表明，凸起处或其附近的残基与南糖构象的偏差非常小。完美双链如预期包含三个A·T碱基对，在H2O NOESY实验中产生三个非常强的T亚氨基-AH2交叉峰。相比之下，使用凸起双链样品在同一实验中仅观察到两个这样的强交叉峰。使用一维NOE对两个T亚氨基峰进行归属，与凸起3'端紧邻的T·A碱基对的破坏一致；这与我们早期在A或T凸起3'端的T处观察到的化学反应性一致。我们还发现了凸起5'端紧邻的G·C碱基对被破坏的证据。
