Designing a novel molecular beacon for surface-immobilized DNA hybridization studies
Designing a novel molecular beacon for surface-immobilized DNA hybridization studies
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DOI：
10.1021/ja9837809
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发表时间：
1999-03-31
影响因子：
15
通讯作者：
Tan, WH
中科院分区：
文献类型：
--
作者：
Fang, XH;Liu, XJ;Tan, WH
我们设计了一种生物素标记的ssDNA分子信标，用于固体界面上的DNA杂交研究。DNA杂交和分子相互作用研究是诊断遗传性疾病的主要工具，其中临床症状与DNA的改变有关。识别人类基因组中的这些突变已经成为许多研究工作的焦点。一个最近的新发展是一类新的寡核苷酸探针，分子信标（MB）。1996年Tyagi和克雷默首次开发的分子信标1是具有茎环结构的单链寡核苷酸探针。分子的环部分可以报告特异性互补核酸的存在。1-5 MB两端的五个碱基彼此互补，形成茎。荧光团和猝灭剂连接到茎的两端，如图1所示。茎保持这两个部分彼此紧密接近，导致荧光团的荧光通过能量转移而猝灭。当探针遇到靶DNA分子时，它形成比茎更长和更稳定的杂交体，并且其刚性和长度排除了茎杂交体的同时存在。因此，MB经历自发的构象重组，迫使茎分开，并导致荧光团和猝灭剂彼此远离，导致荧光恢复。因此，在室温下，MB仅在与其靶分子杂交时才发射强烈的荧光信号。1-8循环的大小及其内容可以通过设计不同的MB来改变。此外，猝灭剂和荧光团可以根据所研究的问题而改变。MB有多种应用，1-8包括聚合酶链反应的实时监测，1甚至HIV-1疾病进展的调查。4、5 MB具有极高的选择性，具有单碱基对错配识别能力。它们在遗传学、疾病机制和分子相互作用的研究中，在疾病诊断和新药开发中的应用中具有很大的前景。可以预期，表面固定化分子信标将有许多有趣的应用。到目前为止，MB仅用于均匀的液体溶液中。这限制了微球在体内生物医学研究和DNA生物传感器开发中的应用。为了充分探索MB的潜力，我们设计了生物素化的ssDNA MB，如图1所示，其旨在固定到二氧化硅表面上用于各种应用。MB由28个碱基组成，其中18个碱基为目的序列，5个碱基对形成茎，以四甲基罗丹明（TMR）为荧光团，DABCYL（dimethylaminoazenzenaminoexal-3-acryinido）为猝灭剂，合成了生物素标记的ssDNA分子信标。DABCYL是一种非荧光发色团，可作为MB中任何荧光团的通用猝灭剂。2 MB设计中有五个重要的考虑因素。首先是表面固定化的官能团。将生物分子固定到固体表面上的最常见方式之一是通过生物素-抗生物素蛋白结合。9，10生物素-抗生物素蛋白与表面的连接适合于DNA杂交。由于5′和3′端分别与荧光团和猝灭剂连接，因此向MB添加生物素官能团是将MB连接到表面的最简单策略。其次是生物素结合的位置。我们尝试了不同的位置来连接生物素：环序列，第二个碱基对的位置...
We have designed a biotinylated ssDNA molecular beacon for DNA hybridization studies at a solid interface. DNA hybridization and molecular interaction studies are major tools for the diagnosis of genetic disease, in which the clinical symptoms are linked to alterations in DNA. Identifying these mutations in human genome has become the focus of many research efforts. One recent new development is a novel class of oligonucleotide probes, molecular beacons (MBs). Molecular beacons, first developed by Tyagi and Kramer in 1996, 1 are single stranded oligonucleotide probes that possess a stem-and-loop structure. The loop portion of the molecule can report the presence of a specific complementary nucleic acid. 1-5 The five bases at the two ends of the MB are complementary to each other, forming the stem. A fluorophore and a quencher are linked to the two ends of the stem, as shown in Figure 1. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When the probe encounters a target DNA molecule, it forms a hybrid that is longer and more stable than the stem, and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the MB undergoes a spontaneous conformational reorganization that forces the stem apart and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence. Therefore, at room temperature, the MBs emit an intense fluorescent signal only when hybridized to their target molecules. 1-8 The size of the loop and its content can be varied by designing different MBs. Also, the quencher and the fluorophores can be changed according to the problem studied. There have been a variety of applications of MBs, 1-8 including the real-time monitoring of polymerase chain reactions, 1 and even the investigation of HIV-1 disease progression. 4, 5 MBs have extremely high selectivity with single base pair mismatch identification capability. They hold great promise for studies in genetics, disease mechanisms, and molecular interactions, for applications in disease diagnostics, and in new drug development. It is expected that there will be many interesting applications for surface-immobilized molecular beacons. So far, MBs have only been used in a homogeneous liquid solution. This limits the applications of MBs in in vivo biomedical studies and in DNA biosensor development. To fully explore the potentials of MBs, we have designed a biotinylated ssDNA MB, shown in Figure 1, which is intended for immobilization onto a silica surface for a variety of applications. The MB has a total of 28 bases, of which 18 bases are the sequence of interest and 5 base pairs form the stem.The biotinylated ssDNA molecular beacon has been synthesized with tetramethylrhodamine (TMR) as the fluorophore and DABCYL (dimethylaminoazobenzen aminoexal-3-acryinido) as the quencher. DABCYL, a nonfluorescent chromophore, serves as a universal quencher for any fluorophore in MBs. 2 There are five important considerations in MB design. First is the functional group for surface immobilization. One of the most common ways for biomolecule immobilization onto a solid surface is through biotin-avidin binding. 9, 10 The biotin-avidin linkage to a surface is suitable for DNA hybridization. Since the 5′ and 3′ ends are linked to a fluorophore and a quencher, respectively, adding a biotin functional group to the MB is the easiest strategy to attach the MB to a surface. Second is the position for biotin binding. We tried different positions to link biotin: the loop sequence, the second base pair position of …