In-Stem-Labeled Molecular Beacons for Distinct Fluorescent Color Readout
In-Stem-Labeled Molecular Beacons for Distinct Fluorescent Color Readout
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DOI：
10.1002/anie.201101968
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Wagenknecht, Hans-Achim
中科院分区：
文献类型：
--
作者：
Holzhauser, Carolin;Wagenknecht, Hans-Achim
分子信标是核酸荧光生物分析研究中广泛使用的工具。[1，2]目前，该领域的最大挑战是监测活细胞中的DNA/RNA摄取和/或序列特异性杂交，以及通过实时聚合酶链反应（PCR）可靠地检测单核苷酸多态性（SNP）。[2]如果在体内进行杂交测定，则由于细胞内组分的自发荧光或不期望的荧光淬灭，单一发射波长的应用具有错误的阳性或错误的阴性读数的风险。因此，需要更好的MB。该领域最重要的发展是无猝灭剂的MB，[3]低噪声无茎PNA MB，[4]波长偏移MB，[5，6]和基于受激准分子荧光颜色读出的MB。[7-11]最近，我们报道了作为DNA中人工核碱基的两个噻唑橙子（TO）发色团形成疏水相互作用的链间二聚体，其导致DNA杂交时荧光颜色的明显变化。[7]在此，我们提出了一种基于TO和噻唑红（TR）的组合作为能量转移（ET）的链间发色团对的茎内标记的波长移动MB的先进设计。使用我们发表的DNA构建块，[12]我们制备了四种MB（DNA 1-DNA 4，图1，表1），其茎长从11个碱基对到5个碱基对（包括作为人工碱基的染料）不等。此外，制备DNA 5以阐明对角TO/TR对（5 '-3'对3 '-5'）的取向的作用。在所有MB中，TO和TR染料嵌入茎中相同的DNA碱基环境中，以便为发色团相互作用提供可比较的结构方案。这包括在发色团对的每一侧上的一个A-T碱基对和作为与每种染料相对的碱基的胸腺嘧啶。除了这个一致的中央部分，茎的顺序是随机的。所有MB的吸收光谱清楚地表明存在两种发色团，在510 nm（TO）和640 nm（TR）处信号分离良好（参见支持信息）。TO/TR MB的功能表征主要通过使用490 nm处的TO选择性激发的稳态荧光光谱来进行。此外，将发夹的解链温度（Tm）与在1.2当量的反链存在下形成的双链体的解链温度（Tm）进行比较。当这些反向链不仅与环区域互补，而且与茎的“内部”部分互补时，它被证明是最佳的。这一结果在使用DNA 2和不同长度的反向链的代表性实验中得到了阐明（参见支持性文献10.2.1）。
Molecular beacons (MBs) are widely used tools in fluorescence bioanalytical studies of nucleic acids.[1, 2] Currently, the biggest challenges in this area are to monitor DNA/RNA uptake and/or sequence-specific hybridization in living cells, and to reliably detect single-nucleotide polymorphism (SNP) by real-time polymerase chain reaction (PCR).[2] If hybridization assays are carried out in vivo, the application of single emission wavelengths bears the risk of wrong positive or wrong negative readout as a result of the autofluorescence of intracellular components or undesired fluorescence quenching. Hence, better MBs are needed. The most important developments in this area have been quencher-free MBs,[3] low-noise stemless PNA MBs,[4] wavelength-shifting MBs,[5, 6] and MBs based on excimer fluorescence color readout.[7–11] Recently, we reported that two thiazole orange (TO) chromophores as artificial nucleobases in DNA form a hydrophobically interacting interstrand dimer which results in a distinct change in fluorescence color upon DNA hybridization.[7] Herein we present an advanced design of in-stemlabeled, wavelength-shifting MBs based on the combination of TO and thiazole red (TR) as an interstrand chromophore pair for energy transfer (ET).Using our published DNA building blocks,[12] we prepared four MBs (DNA1–DNA4, Figure 1, Table 1), which vary in stem length from 11 down to 5 base pairs (including the dyes as artificial bases). In addition DNA5 was prepared to elucidate the role of the orientation of the diagonal TO/TR pair (5’–3’vs. 3’–5’). In all MBs, the TO and TR dyes were embedded in an identical DNA base environment in the stem in order to provide comparable structural scenarios for the chromophore interactions. This includes one A–T base pair on each side of the chromophore pair and thymines as the bases opposite to each dye. Except for this consistent central part, the sequence of the stem is random. The absorption spectra of all MBs clearly show the presence of both chromophores with well-separated signals at 510 nm (TO) and 640 nm (TR)(see the Supporting Information). The functional characterization of the TO/TR MBs was performed mainly by steady-state fluorescence spectroscopy using the TO-selective excitation at 490 nm. Additionally the melting temperatures (Tm) of the hairpins were compared with those of the duplexes formed in the presence of 1.2 equiv of counterstrands. It proved to be optimal when these counterstrands were complementary not only to the loop region but also to the “inner” parts of the stem. This result was elucidated in representative experiments with DNA2 and counterstrands of different lengths (see the Supporting