Steady-state and time-resolved fluorescence studies indicate an unusual conformation of 2-aminopurine within ATAT and TATA duplex DNA sequences
Steady-state and time-resolved fluorescence studies indicate an unusual conformation of 2-aminopurine within ATAT and TATA duplex DNA sequences
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DOI：
10.1093/nar/gkg339
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发表时间：
2003-05-01
影响因子：
14.9
通讯作者：
Linn, S
中科院分区：
文献类型：
--
作者：
Rai, P;Cole, TD;Linn, S
2-氨基嘌呤 (2-AP) 是腺嘌呤的荧光类似物，已被广泛用作局部 DNA 构象的探针，因为 2-AP 的激发和发射特性以及荧光寿命在 DNA 内以序列依赖性方式变化。使用稳态和时间分辨荧光技术，我们发现 2-AP 似乎异常地堆积在双链 DNA 中 ATAT 和 TATA 的内部位置。在这些背景下 2-AP 的激发波长最大值发生红移，表明荧光团的溶剂暴露减少。此外，在这些情况下，2-AP 荧光对丙烯酰胺依赖性碰撞猝灭具有抵抗力，这表明荧光团受到其在双链体中的堆叠位置的保护。荧光激发光谱中 275 nm 处的次级峰的存在进一步证实了这一结论，该次级峰表明附近非荧光 DNA 碱基的激发能量转移有效。在这些交替的 AT 环境中，2-AP 的荧光各向异性衰减和内部角度“摆动”运动测量也与荧光团在基堆栈内受到高度约束和固定一致。当这些荧光特征与其他双链 DNA 序列背景下的 2-AP 荧光特征进行比较时，它们是独一无二的。
2-Aminopurine (2-AP), a fluorescent analog of adenine, has been widely used as a probe for local DNA conformation, since excitation and emission characteristics and fluoresence lifetimes of 2-AP vary in a sequence-dependent manner within DNA. Using steady-state and time-resolved fluorescence techniques, we report that 2-AP appears to be unusually stacked in the internal positions of ATAT and TATA in duplex DNA. The excitation wavelength maxima for 2-AP within these contexts were red shifted, indicating reduced solvent exposure for the fluorophore. Furthermore, in these contexts, 2-AP fluorescence was resistant to acrylamide-dependent collisional quenching, suggesting that the fluorophore is protected by its stacked position within the duplex. This conclusion was further reinforced by the presence of a secondary peak at 275 nm in the fluorescence excitation spectra that is indicative of efficient excitation energy transfer from nearby non-fluorescent DNA bases. Fluorescence anisotropy decay and internal angular 'wobbling' motion measurements of 2-AP within these alternating AT contexts were also consistent with the fluorophore being highly constrained and immobile within the base stack. When these fluorescence characteristics are compared with those of 2-AP within other duplex DNA sequence contexts, they are unique.