Low-Noise Stemless PNA Beacons for Sensitive DNA and RNA Detection
Low-Noise Stemless PNA Beacons for Sensitive DNA and RNA Detection
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DOI：
10.1002/anie.200803549
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Seitz, Oliver
中科院分区：
文献类型：
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作者：
Socher, Elke;Bethge, Lucas;Seitz, Oliver
在各种生物测定中需要发出特定核酸存在信号的荧光探针，包括DNA定量、SNP分型（SNP=单核苷酸多态性）和活细胞中mRNA表达的分析。[1]大多数探针利用两个发色团之间的距离依赖性相互作用。灵敏的荧光杂交探针显示大的hybridizationinduced增强的荧光发射，这可能会达到信号与背景比（SBR）的顺序为102。[2]选择性探针能够实现单核苷酸特异性荧光信号传导。使用DNA分子信标（MB，方案1A）在灵敏和特异的DNA和RNA检测中均取得了成功。[3]这些发夹形状的探针被设计成使两种相互作用的染料非常接近。SBR是高的，因为在不存在靶标的情况下，荧光通过荧光共振能量转移（FRET）、碰撞猝灭和/或基态或激发态复合物的形成而被有效地猝灭。分子信标以高匹配/错配特异性结合靶DNA，但仅在一定的温度范围内，该温度范围取决于匹配和错配的探针-靶复合物的热稳定性之间的差异。[4]因此，在匹配和错配的探针-靶标复合物共存的条件下，不可能区分匹配的靶标和错配的靶标。分子信标设计的主要限制是增加灵敏度的特征对荧光信号的序列特异性是有害的，反之亦然。只有当茎区域容易打开时才能获得大的荧光增强，而高特异性要求稳定的茎抵抗通过错配杂交打开。我们设想了另一种信标设计。该方法一方面保留了分子信标中使用的信号传导机制，其中两个发色团检测探针构象的变化，但它省略了形成稳定发夹结构的要求。[5]另一方面，使用“智能”标记，其仅当供体染料嵌入完全匹配的碱基对中时才变为荧光并启动FRET至近红外染料。结果表明，这两个过程的组合，检测构象变化的能量转移机制和信号的转换的嵌入染料的改变堆叠相互作用，允许高达108倍的荧光增强杂交后。重要的是，无茎探针在几乎任何温度下都能区分匹配和不匹配的目标。证明了DNA和RNA靶标的均质检测。设计方法如方案1B所示。嵌入剂染料，如噻唑橙子，被引入作为基于肽核酸（PNA）的探针中的碱基替代物，并用作FRET的供体。末端附加的近红外（NIR）染料，如NIR 667，用作受体染料。预期单链探针中供体的激发将诱导受体染料的可忽略的发射，因为1）供体激发态由于围绕未堆叠的噻唑橙子的中心次甲基桥的扭转运动而迅速耗尽，[6] 2）NIR 667（受体）染料在与核碱基碰撞时淬灭，和3）分子内染料-染料二聚体或短寿命碰撞复合物可形成，这得益于不带电荷的疏水PNA分子采用方案1中的塌陷结构的趋势。A）分子信标与B）无茎FIT-PNA信标在互补核酸检测中的比较。在...
Fluorescent probes that signal the presence of specific nucleic acids are required in a variety of bioassays, including DNA quantification, SNP typing (SNP= single-nucleotide polymorphism), and analysis of mRNA expression in living cells.[1] The majority of probes take advantage of the distancedependent interaction between two chromophores. Sensitive fluorescent hybridization probes show large hybridizationinduced enhancements of fluorescence emission, which may reach signal-to-background ratios (SBR) on the order of 102.[2] Selective probes enable single-nucleotide-specific fluorescence signaling. Success in both sensitive and specific DNA and RNA detection has been achieved using DNA molecular beacons (MBs, Scheme 1A).[3] These hairpin-shaped probes have been designed to bring the two interacting dyes into close proximity. The SBR is high, because in the absence of target the fluorescence is efficiently quenched by fluorescence resonance energy transfer (FRET), collisional quenching, and/or formation of ground-or excited-state complexes. Molecular beacons bind target DNA with high match/mismatch specificity, but only within a certain temperature range that depends on the difference between thermal stabilities of matched and mismatched probe–target complexes.[4] It is, thus, impossible to distinguish matched from mismatched targets at conditions for which both matched and mismatched probe–target complexes co-exist. The major limitation in molecular-beacon design is that features that increase sensitivity are detrimental to the sequence specificity of fluorescence signaling and vice versa. Large fluorescence enhancements can only be obtained when the stem region is readily opened, while high specificity calls for stable stems that resist opening by mismatched hybridization. We envisioned an alternative beacon design. The approach on one hand retains a signaling mechanism used in molecular beacons, wherein two chromophores detect changes of probe conformation, but it omits the requirement for the formation of stable hairpin structures.[5] On the other hand,“smart” labels are used that become fluorescent and initiate FRET to a near-infrared dye only when the donor dye is embedded in perfectly matched base pairs. It is shown that the combination of the two processes, detection of conformational changes by a switch in energy transfer mechanisms and signaling of altered stacking interactions of an intercalator dye, allows for up to 108-fold fluorescence intensification upon hybridization. Importantly, the stemless probes distinguish matched from mismatched targets at virtually any temperature. Homogeneous detection of both DNA and RNA targets is demonstrated. The design approach is illustrated in Scheme1B. An intercalator dye, such as thiazole orange, is introduced as base surrogate in a peptide nucleic acid (PNA)-based probe and used as donor for FRET. A terminally appended nearinfrared (NIR) dye, such as NIR667, serves as acceptor dye. It was expected that excitation of the donor in single-stranded probes would induce negligible emission of the acceptor dye because 1) the donor excited state is rapidly depleted owing to torsional motion around the central methine bridge of unstacked thiazole orange,[6] 2) the NIR667 (acceptor) dye is quenched upon collisions with nucleobases, and 3) intramolecular dye–dye dimers or short-lived collision complexes may form, aided by the tendency of the uncharged, hydrophobic PNA molecule to adopt a collapsed structure inScheme 1. Comparison of A) molecular beacons with B) stemless FIT–PNA beacons in the detection of complementary nucleic acids. In …