Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland.
Anal Bioanal Chem. 2011 Mar;399(9):3157-76. doi: 10.1007/s00216-010-4304-5. Epub 2010 Oct 29.
It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays.
众所周知，核酸在生物体中发挥着重要作用，因为它们储存和传递遗传信息，并利用这些信息来指导蛋白质的合成。然而，人们对核酸结合特定配体的能力以及寡核苷酸作为分子探针或生物传感器的应用了解较少。寡核苷酸探针是单链核酸片段，可以根据不同的靶标（包括核酸、蛋白质、小分子和离子）进行定制，具有高度的特异性和亲和力。可以将基于寡核苷酸的探针分为两大类：基于形成互补碱基对的杂交探针，以及利用非核酸分析物选择性识别的适体探针，可以与免疫传感器进行比较。杂交和适体探针的设计和构建相似。通常，通过共价连接报告基团（荧光探针中的一个或多个荧光团）来修饰具有预定碱基序列和长度的寡核苷酸（DNA、RNA）。荧光标记作为传感器，将生物识别（杂交、配体结合）转化为荧光信号。荧光标记具有几个优点，例如高灵敏度和多种转换方法（荧光猝灭或增强、荧光各向异性、荧光寿命、荧光共振能量转移（FRET）和激基-单体光开关）。这些多重信号选择与识别元件（DNA、RNA、PNA、LNA）的设计灵活性和各种标记策略相结合，有助于开发许多选择性和灵敏的生物测定法。本综述涵盖了寡核苷酸探针的设计和工程基础，描述了典型的构建方法，并讨论了在杂交研究和适体基测定中使用的探针示例。
