Rupcich Nicholas, Chiuman William, Nutiu Razvan, Mei Shirley, Flora Kulwinder K, Li Yingfu, Brennan John D
Department of Chemistry, McMaster University, Hamilton, Ontario, Canada L8S 4M1.
J Am Chem Soc. 2006 Jan 25;128(3):780-90. doi: 10.1021/ja053336n.
Recent years have seen a dramatic increase in the use of fluorescence-signaling DNA aptamers and deoxyribozymes as novel biosensing moieties. Many of these functional single-stranded DNA molecules are either engineered to function in the presence of divalent metal ion cofactors or designed as sensors for specific divalent metal ions. However, many divalent metal ions are potent fluorescence quenchers. In this study, we first set out to examine the factors that contribute to quenching of DNA-bound fluorophores by commonly used divalent metal ions, with the goal of establishing general principles that can guide future exploitation of fluorescence-signaling DNA aptamers and deoxyribozymes as biosensing probes. We then extended these studies to examine the effect of specific metals on the signaling performance of both a structure-switching signaling DNA aptamer and an RNA-cleaving and fluorescence-signaling deoxyribozyme. These studies showed extensive quenching was obtained when using divalent transition metal ions owing to direct DNA-metal ion interactions, leading to combined static and dynamic quenching. The extent of quenching was dependent on the type of metal ion and the concentration of supporting monovalent cations in the buffer, with quenching increasing with the number of unpaired electrons in the metal ion and decreasing with the concentration of monovalent ions. The extent of quenching was independent of the fluorophore, indicating that quenching cannot be alleviated simply by changing the nature of the fluorescent probe. Our results also show that the DNA sequence and the local secondary structure in the region of the fluorescent tag can dramatically influence the degree of quenching by divalent transition metal ions. In particular, the extent of quenching is predominantly determined by the fluorophore location with respect to guanine-rich and duplex regions within the strand sequence. Examination of the effect of both the type and concentration of metal ions on the performance of a fluorescence-signaling aptamer and a signaling deoxyribozyme confirms that judicious choice of divalent transition metal ions is important in maximizing signals obtained from such systems.
近年来，荧光信号DNA适配体和脱氧核酶作为新型生物传感部分的应用急剧增加。许多这些功能性单链DNA分子要么被设计成在二价金属离子辅因子存在下起作用，要么被设计成特定二价金属离子的传感器。然而，许多二价金属离子是有效的荧光猝灭剂。在本研究中，我们首先着手研究导致常用二价金属离子猝灭与DNA结合的荧光团的因素，目的是建立能够指导未来将荧光信号DNA适配体和脱氧核酶用作生物传感探针的一般原则。然后，我们扩展了这些研究，以检查特定金属对结构转换信号DNA适配体和RNA切割及荧光信号脱氧核酶的信号传导性能的影响。这些研究表明，由于直接的DNA-金属离子相互作用，使用二价过渡金属离子时会发生广泛的猝灭，导致静态和动态猝灭的结合。猝灭程度取决于金属离子的类型和缓冲液中支持单价阳离子的浓度，猝灭随着金属离子中未配对电子的数量增加而增加，随着单价离子的浓度降低而降低。猝灭程度与荧光团无关，这表明不能简单地通过改变荧光探针的性质来减轻猝灭。我们的结果还表明，荧光标签区域内的DNA序列和局部二级结构可以显著影响二价过渡金属离子的猝灭程度。特别是，猝灭程度主要由荧光团相对于链序列中富含鸟嘌呤和双链区域的位置决定。检查金属离子的类型和浓度对荧光信号适配体和信号脱氧核酶性能的影响证实，明智地选择二价过渡金属离子对于最大化从此类系统获得的信号很重要。
