Target-catalyzed transfer reactions for the amplified detection of RNA
Target-catalyzed transfer reactions for the amplified detection of RNA
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DOI：
10.1002/anie.200801355
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Seitz, Oliver
中科院分区：
文献类型：
--
作者：
Grossmann, Tom N.;Roeglin, Lars;Seitz, Oliver
Signal amplification is key to the detection of small amounts of biological targets. In nucleic acid diagnostics the polymerase chain reaction (PCR) is the standard method for highly sensitive detection of DNA and RNA. The PCR first amplifies the target and allows the subsequent detection of formed product. Detection methods that do not require amplification of the target could lead to simpler devices and are highly desired. PCR-less detection has been approached by taking advantage of the catalyzed formation of signaling molecules [1] as well as by electrochemical [2] and nanoparticlebased assays.[3] High sensitivities have been obtained by the coupling of two amplification methods. For example, in a barcode-based method one molecule of target DNA first recruits multiple copies of DNA tags, which are detected in a second step by means of nanoparticle-catalyzed development of silver.[4] A preamplification step was also included in a sensitive peptide nucleic acid (PNA)-based hybridization assay wherein one target DNA acquired multiple copies of horseradish peroxidase for inducing the catalytic production of dye molecules.[1b] The recruiting step typically involves binding of the target molecule to an immobilized capture oligonucleotide and a second detector oligonucleotide probe that features traceable entities (eg bar-code DNA or biotin) for detection of complementary sequences. However, the noncovalent nature of the formed sandwich complexes sets limits on the stringency of washing protocols required to remove nonbound detection oligonucleotides. These sandwich assays draw upon the recruitment of a catalytically active moiety to a specific DNA structure. In an alternative design, the target itself may act as a catalyst of a chemical reaction, for example, by aligning the functional groups of modified oligonucleotides.[5–8] This alignment allows the acceleration of reactions such as ligations [5, 9] and hydrolyses [6] which proceed very slowly in the absence of the template. The previous DNA-templated reactions have met these criteria with only limited success. DNA-catalyzed reactions have allowed up to 102-fold signal amplification, which is not sufficient to achieve PCR-less detection of small amounts of DNA. Herein, we introduce target-catalyzed reactions for preamplification of signals that can passed on to enzyme-based detection platforms. The reaction system was designed such that the target catalyzes the covalent attachment of detectable groups to nucleic acid based probes. We show that the preamplification and the covalent mode of reporter-group attachment allow increases of the sensitivity of an enzyme-linked immunosorbant assay (ELISA)-derived detection method.Recently, we introduced the target-catalyzed transfer of a reporter group R from a donating PNA probe 1 to an accepting PNA probe 2 (Figure 1A) as a concept for the detection of nucleic acid targets. Probes 1 and 2 are designed to anneal adjacently to complementary segments of the target. The alignment of thioester 1 and the N-terminal isocysteine (iCys) 2 in the ternary complex target· 1· 2 facilitates a thiol-exchange reaction. By analogy to native chemical ligation [10] the formed thioester intermediate 4 spontaneously reacts by means of an irreversible S! N acyl migration yielding product 5. Reactant and product probes have similar
