Target-catalyzed hairpin structure-mediated padlock cyclization for ultrasensitive rolling circle amplification

滚动圆复制 化学 分子信标 DNA 连接酶连锁反应 脱氧核酶 分子生物学 DNA连接酶 底漆(化妆品) 基因 组合化学 杂交探针 分子探针 聚合酶链反应 寡核苷酸 聚合酶 生物化学 生物 多重聚合酶链反应 有机化学
作者
Huanxia Song,Zhe Yang,Min Jiang,Guo Zhang,Yansha Gao,Zhifa Shen,Zai‐Sheng Wu,Yongliang Lou
出处
期刊:Talanta [Elsevier]
卷期号:204: 29-35 被引量:12
标识
DOI:10.1016/j.talanta.2019.05.057
摘要

Because STAT3 is a potent proto-oncogene, screening STAT3 gene has potential for use in tumor diagnosis, classification of subtypes, and molecular target therapy. Thus, in this study, using STAT3 gene as the model molecule, we developed a novel amplification strategy, ultrasensitive rolling circle amplification (THP-RCA) based on target-catalyzed hairpin structure-mediated padlock cyclization, for the ultrasensitive detection of human proto-oncogenes in a homogenous solution. In this system, HP1 was designed as the cyclization template and RCA reaction primer, while HP2 was the padlock probe. The two probes can fold into a hairpin structure via the self-hybridization and thus lock the signaling process in the absence of target species. The hybridization of HP2 with HP1 in an end-to-end fashion occurs with the help of target DNA. Subsequently, HP2 is cyclized by ligase on HP1 template. Interestingly, during the hybridization and enzymatic cyclization of HP2, the target DNA only serves as the catalytic probe and is not exhausted. The cyclized HP2 enables the rolling circle amplification, generating a long tandem single-stranded (ss) DNA product that is capable of hybridizing with considerable quantity of molecular beacons (MBs). As a result, the dramatically amplified fluorescence value is achieved for the ultrasensitive detection of the STAT3 gene. As a result, target DNA is able to be quantified down to 100 fM with a high specificity towards wild-type target DNA. Moreover, the sensing system is suitable for the target detection in human serum. The novel sensing strategy shows tremendous prospect for application in tumor diagnosis and clinical therapy guidance.
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