A Novel Label‐Free Biosensor Using an Aptazyme–Suppressor‐tRNA Conjugate and an Amber Mutated Reporter Gene
A Novel Label‐Free Biosensor Using an Aptazyme–Suppressor‐tRNA Conjugate and an Amber Mutated Reporter Gene
复制标题
使用适体酶-抑制子-tRNA 缀合物和 Amber 突变报告基因的新型无标记生物传感器
作者：
A. Ogawa;M. Maeda
DNA或RNA适体由于其高特异性的靶结合能力而具有作为检测靶分子的生物传感器的巨大潜力。适体-靶标复合物的形成可以通过质量敏感检测技术，如表面等离子体共振（SPR）或石英晶体微天平（QCM），或通过荧光、电化学、比色或浊度法，通过使用适当标记的aptaACHTUNGTRENNUNG聚体或探针来分析。由适体和核酶组成的适体酶可以将适体-靶标复合物形成的信号转化为核酶活性的信号，因此它们作为基于适体的传感器也引起了关注。然而，这些核酶信号的检测也需要特殊的检测器和/或核酶的标记，与上述适体传感器的情况非常相似。另一方面，我们最近开发了一种基于适体酶的riboACHTUNGTRENNUNG开关作为一种无标记和无检测器的生物传感器。在这种riboACHTUNGTRENNUNG开关传感器中，在原核无细胞翻译系统的帮助下，适体-靶复合物形成信号被转化为易于检测的信号，即下游报告蛋白的表达。由于报告蛋白的表达信号可以容易地检测到，在某些情况下可以明显地检测到，因此不需要标记适体酶或特殊的检测器。此外，使用翻译系统的好处不仅在于无标记和无检测器的传感，而且还在于其在体内调节基因表达的适用性。我们在此报告了一种新型的无标记和适体酶为基础的生物传感器，它使用的翻译系统与无义密码子抑制方法相结合，是不同于以前开发的核糖开关生物传感器。无义密码子抑制方法是将天然或非天然氨基酸分配给mRNA上的无义密码子（琥珀、蛋白石或赭石密码子）的方法。核糖体通常在无义密码子处终止，合成的蛋白质通过释放因子（RF）的作用从mRNA中去除。在原核翻译系统中，有三个RF，其中两个直接识别无义密码子;释放因子1（RF 1）识别琥珀和赭石密码子，释放因子2（RF 2）识别蛋白石和赭石密码子。然而，当存在氨酰化的抗ACHTUNGTRENNUNG密码子调节的抑制性tRNA时，抑制性tRNA与RF竞争掺入mRNA无义密码子上的核糖体中。如果抑制型tRNA赢得了这场竞争，翻译就会继续（即无义密码子被抑制），但如果RFs获胜，翻译就会停止。在不存在RF或存在RF抑制剂的情况下，抑制效率变得高得多。最近，Ogawa及其同事使用了这种无义抑制方法，在ACHTUNGTRENNUNG构建的原核无细胞翻译系统中，在没有RF的情况下，使用了三种抑制性tRNA，这三种抑制性tRNA对应于三种无义密码子中的每一种;这使得核糖体能够读取所有无义密码子。这些抑制性tRNA是大肠杆菌的衍生物。大肠杆菌的tRNA被Ser-tRNA合成酶（SerRS）识别，并迅速地装载Ser，即使它们的反密码子被改变为另一个反密码子，并且不使用修饰的碱基。在这项研究中，我们将茶碱依赖性适体酶连接到反密码子调节的抑制剂tRNACUA的5'末端以获得琥珀密码子，[11]并将适体酶-抑制剂-tRNA缀合物（AST）与翻译系统中的琥珀突变报告基因（荧光素酶）组合以构建新型基于适体酶的生物传感器。本研究中基于适体酶的生物传感器的基本概念如图1所示。该生物传感器系统由不含RF 1的重组原核无细胞翻译系统和两种RNA分子组成：AST（图1，左上）和琥珀突变报告基因（图1，左下）。在缺乏适体酶的靶点的情况下（图1，左;关闭状态），预期AST不会被氨酰化ACHTUNGTRENNUNG，因为氨酰tRNA合成酶（ARS）对tRNA的受体茎的结构敏感。在这种情况下，也就是说，在缺乏“激活的”抑制tRNA的情况下，由于RF 1的缺乏，核糖体在报告基因上的琥珀密码子处停滞。另一方面，当靶标与AST的适体酶结合时（图1，右; ON状态），它诱导适体酶的自切割;这产生“活化的”抑制tRNA，其可以被相应的ARS识别并被相应的氨基酸氨酰化。这种氨酰化的抑制性tRNA在琥珀密码子处被掺入停止的核糖体中;这使得核糖体继续合成报告蛋白，直到它到达由RF 2识别的“真正”末端密码子（赭石密码子：UAA）。因此，全长活性报告蛋白的表达是靶分子存在的指示。为了首先研究在其5'末端具有额外序列的抑制性tRNA是否被ARS识别并被aminoACHTUNGTRENNUNG酰化，我们制备了两种不同类型的抑制性tRNA和两种不同类型的编码荧光素酶基因的mRNA的四种DNA模板（图2A和2B）。其中一个抑制性tRNA是正常的抑制性tRNACUA（supT），另一个（5SL-supT）与supT相同，除了在其5'末端具有额外的茎环序列。两个luci[a] Dr. A.小川博士Maeda Bioengineering Laboratory RIKEN（The Institute of Physical and Chemical Research）2-1 Hirosawa，和子，琦玉351-0198（Japan）传真：（+81）48 -462-4658 E-mail：a-ogawa@riken.jp本文的支持信息可在http://www.chembiochem.org下的WWW上获得或从作者处获得。
DNA or RNA aptamers have great potential as biosensors for detecting target molecules owing to their high and specific target-binding ability. Aptamer–target complex formation can be analyzed by mass-sensitive detection techniques, such as surface plasmon resonance (SPR) or quartz crystal microbalance (QCM), or by fluorescent, electrochemical, colorimetric, or turbidimetric methods by using appropriately labeled aptaACHTUNGTRENNUNGmers or probes. Aptazymes, which are composed of an aptamer and a ribozyme, can convert the signal of the aptamer– target complex formation to that of ribozyme activity, so that they have also garnered attention as aptamer-based sensors. Nevertheless, detection of these ribozyme signals also requires special detectors and/or labeling of ribozymes, much as in the case of the aptamer sensors described above. On the other hand, we have recently developed an aptazyme-based riboACHTUNGTRENNUNGswitch as a label-free and detector-free biosensor. In this riboACHTUNGTRENNUNGswitch sensor, the aptamer–target complex formation signal is converted to an easily detectable signal—that is, expression of a downstream reporter protein—with the help of a prokaryotic cell-free translation system. As the expression signal of the reporter protein can be detected easily, in some cases visibly, labeling of aptazymes or special detectors is not required. Moreover, the benefits of using the translation system lie not only in the labeland detector-free sensing, but also to its applicability to regulating gene expression in vivo. We report herein a new type of label-free and aptazyme-based biosensor that uses a translation system in combination with a nonsense codon suppression method, and is distinct from the riboswitch biosensor developed previously. The nonsense codon suppression method is a method by which a natural or unnatural amino acid is assigned to the nonsense codon (amber, opal, or ochre codon) on the mRNA. The ribosome usually stops at the nonsense codon, and the synthesized protein is removed from the mRNA by the action of release factors (RFs). In a prokaryotic translation system, there are three RFs and two of these directly recognize the nonsense codons; release factor 1 (RF1) recognizes the amber and ochre codons and release factor 2 (RF2) recognizes the opal and ochre codons. However, in the presence of an antiACHTUNGTRENNUNGcodon-adjusted suppressor tRNA, which is aminoacylated, the suppressor tRNA competes with the RFs for being incorporated into the ribosome on the nonsense codon of the mRNA. If the suppressor tRNA wins this competition, translation continues (i.e. , the nonsense codon is suppressed) but if the RFs win, translation stops. In the absence of RFs or in the presence of inhibitors for RFs, the suppression efficiency becomes much higher. Recently, Ogawa and co-workers used this nonsense suppression method with the three kinds of suppressor tRNA that correspond to each of the three nonsense codons in a ACHTUNGTRENNUNGreconstituted prokaryotic cell-free translation system in the absence of RFs; this enabled the ribosome to read through all nonsense codons. These suppressor tRNAs were derivatives of E. coli tRNA, which were recognized by Ser-tRNA synthetase (SerRS) and rapidly charged with Ser, even if their anticodon was changed into another anticodon and no modified base was used. In this study, we tethered a theophylline-dependent aptazyme to the 5’ terminus of the anticodon-adjusted suppressor tRNACUA for an amber codon, [11] and combined the aptazyme–suppressor-tRNA conjugate (AST) with an amber-mutated reporter gene (luciferase) in the translation system to construct a novel aptazyme-based biosensor. The basic concept of the aptazyme-based biosensor in this study is illustrated in Figure 1. This biosensor system consists of an RF1-free reconstituted prokaryotic cell-free translation system and two RNA molecules: an AST (Figure 1, upper left) and an amber-mutated reporter gene (Figure 1, lower left). In the absence of the target of the aptazyme (Figure 1, left ; OFF state) it is expected that AST will not be aminoacylated ACHTUNGTRENNUNGbecause aminoacyl tRNA synthetase (ARS) is sensitive to the structure of an accepter stem of tRNA. Under this condition, that is, in the absence of an “activated” suppressor tRNA, the ribosome stalls at the amber codon on the reporter gene because of the absence of RF1. On the other hand, when the target binds to the aptazyme of AST (Figure 1, right; ON state) it induces self-cleavage of the aptazyme; this produces the “activated” suppressor tRNA, which can be recognized by the corresponding ARS and aminoacylated with the corresponding amino acid. This aminoacylated suppressor tRNA is incorporated into the stalling ribosome at the amber codon; this causes the ribosome to continue with the synthesis of the reporter protein until it reaches the “true” terminal codon (ochre codon: UAA), which is recognized by RF2. Therefore, the expression of a full-length active reporter protein is an indicator of the existence of the target molecule. To first investigate whether or not a suppressor tRNA with an extra sequence at its 5’ terminus is recognized and aminoACHTUNGTRENNUNGacylated by ARS, we prepared four DNA templates for two different kinds of suppressor tRNAs and two different kinds of mRNAs that coded luciferase genes (Figure 2A and 2B). One of the suppressor tRNAs is a normal suppressor tRNACUA (supT) and the other (5SL-supT) is identical to supT except that it has an extra stem–loop sequence at its 5’ terminus. The two luci[a] Dr. A. Ogawa, Dr. M. Maeda Bioengineering Laboratory RIKEN (The Institute of Physical and Chemical Research) 2–1 Hirosawa, Wako, Saitama 351–0198 (Japan) Fax: (+81)48-462-4658 E-mail : a-ogawa@riken.jp Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.
影响因子：
14.9
作者：
SAMPSON, JR;SAKS, ME
通讯作者：
SAKS, ME
影响因子：
7.4
作者：
Li, Yuan;Lee, Hye Jin;Corn, Robert M.
通讯作者：
Corn, Robert M.
影响因子：
4.5
作者：
Sekella, PT;Rueda, D;Walter, NG
通讯作者：
Walter, NG