Pyrene-Excimer Probes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acids in Complex Biological Fluids
Pyrene-Excimer Probes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acids in Complex Biological Fluids
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基于杂交链式反应的芘-准分子探针用于检测复杂生物液体中的核酸
DOI：
10.1002/anie.201005375
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Tan, Weihong
中科院分区：
文献类型：
--
作者：
Huang, Jin;Wu, Yanrong;Tan, Weihong
核酸的灵敏和选择性检测在生物学研究、临床诊断和生物防御应用中是重要的。由于感兴趣的DNA序列可能以非常小的量存在，因此有必要开发能够检测痕量水平的特定序列的扩增技术。现有的DNA扩增技术可以分为两大类：热循环和等温处理。聚合酶链反应（PCR）是用于DNA扩增的最广泛使用的热循环方案。[1]反应以指数方式进行，因此痕量的DNA可以被扩增到可检测的水平。另一种热循环方法是连接酶链式反应（LCR）。[2]因为热稳定连接酶在多次热循环后保持活性，所以来自一轮的连接产物可以成为下一轮连接的靶标。以这种方式，产品的量可以通过重复的热循环以指数方式增加。然而，热循环方法是耗时的，有时是非特异性的，并且仅限于热稳定酶和实验室环境。在等温扩增的情况下，链置换聚合酶通常用于DNA双链体的一条链的连续复制。例如，在滚环扩增（RCA）中，环状寡核苷酸序列作为模板，用于创建包含环状寡核苷酸编码序列的周期性重复的互补单链DNA链。[3]另一种技术，链置换扩增（SDA），它是基于聚合或断裂，[4，5]是利用在几个敏感的DNA检测方法。[6-8]DNA扩增的进一步发展是杂交链反应（HCR），其中两种稳定的DNA发夹物种在溶液中共存，直到引入引发剂链。引发剂触发级联的杂交事件，以产生类似于交替共聚物的带切口的双螺旋。[9-11]复杂生物液体中核酸的定量是生物医学应用的另一个挑战，主要是因为探针和生物液体都观察到背景信号。然而，通过引入芘部分，可以有效地解决该问题。[12-14]最近，芘已被用作荧光染料，以指示离子，[15]小分子，[16，17]核酸，[12，14]或蛋白质的存在。[13芘用作空间敏感的荧光染料。当激发态分子与基态的芘部分紧密接近时，可以形成激基缔合物。激基缔合物的形成导致发射（对于单体，从375和398 nm）向更长波长（485 nm）偏移。激基缔合物在485 nm波长处的寿命（高达100 ns）也比生物流体中的发色团（小于10 ns）长。[12-14]受激准分子的长寿命为使用该探针进行生物介质中的靶检测提供了机会，因为时间分辨荧光测量应该能够去除强细胞背景信号以进行有效分析。在此，我们描述了一种DNA检测系统，该系统将HCR的扩增能力与结合到发夹探针的芘分子的空间敏感荧光信号相结合（图1）。DNA发夹H1* 和H2* 通过每个末端的六个碳原子间隔区用芘部分双标记。每个发夹具有包围6个核苷酸（nt）环的18个碱基对的茎。每一个在H1* 的5о端也有一个额外的6 nt粘性末端（与H1 * 的环互补）。
The sensitive and selective detection of nucleic acids is important in biological studies, clinical diagnostics, and biodefense applications. Since the DNA sequences of interest may be present in very small amounts, it is necessary to develop amplification techniques that enable the detection of trace levels of a specific sequence. Existing DNA-amplification techniques can be divided into two broad categories: thermal cycling and isothermal processing. The polymerase chain reaction (PCR) is the most widely used thermal-cycling protocol for DNA amplification.[1] The reaction proceeds exponentially, so that trace amounts of DNA can be amplified to detectable levels. Another thermalcycling method is the ligase chain reaction (LCR).[2] Because a thermostable ligase retains activity after multiple thermal cycles, the ligation products from one round can become the targets for the next round of ligation. In this way, the amount of product can increase in an exponential way by repeated thermal cycling. However, thermal-cycling methods are timeconsuming, sometimes nonspecific, and limited to a thermostable enzyme and a laboratory setting. In the case of isothermal amplification, a strand-displacement polymerase is often used for the continuous replication of one strand of a DNA duplex. For example, in rolling circle amplification (RCA), a circular oligonucleotide sequence serves as a template for the creation of a complementary single-stranded DNA chain containing periodic repeats of the sequence coded by the circular oligonucleotide.[3] Another technique, strand-displacement amplification (SDA), which is based on polymerization or scission,[4, 5] is utilized in several sensitive DNA-detection methods.[6–8] A further development in DNA amplification is the hybridization chain reaction (HCR), in which two stable species of DNA hairpins coexist in solution until an initiator strand is introduced. The initiator triggers a cascade of hybridization events to yield nicked double helices analogous to alternating copolymers.[9–11] The quantitation of nucleic acids in complex biological fluids is another challenge for biomedical applications, essentially because of the background signals observed for both the probe and biological fluids. However, by the introduction of pyrene moieties, the problem can be addressed effectively.[12–14] Recently, pyrene has been used as a fluorescent dye to signal the presence of ions,[15] small molecules,[16, 17] nucleic acids,[12, 14] or proteins.[13, 18] Pyrene acts as a spatially sensitive fluorescent dye. An excimer can form when an excited-state molecule is brought into close proximity with a pyrene moiety in the ground state. The formation of the excimer results in a shift of the emission (from 375 and 398 nm for the monomer) to a longer wavelength (485 nm). The excimer also has a longer lifetime at the wavelength of 485 nm (up to 100 ns) than chromophores in biological fluids (less than 10ns).[12–14] The long lifetime of the excimer provides an opportunity for the use of this probe for target detection in biological media, as time-resolved fluorescence measurements should enable the removal of the strong cellular background signal for efficient analysis. Herein we describe a DNA-detection system which combines the amplification capability of HCR with the spatially sensitive fluorescence signal of pyrene molecules conjugated to hairpin probes (Figure 1). The DNA hairpins H1* and H2* are dual-labeled with pyrene moieties through a six-carbon-atom spacer at each end. Each hairpin has a stem of 18 base pairs enclosing a 6 nucleotide (nt) loop. Each also has an additional 6nt sticky end at the 5о end of H1*(complementary to the loop of …