高等学校化学学报

高等学校化学学报

• 研究论文 • 上一篇    

有机溶剂体系中刺激-响应性RNA切割型脱氧核酶的工程化

贺荣荣，游昕，郑星，杨梦晗，常天俊   

  1. 河南理工大学资源环境学院
  • 收稿日期:2025-01-09 修回日期:2025-02-16 网络首发:2025-02-20 发布日期:2025-02-20
  • 通讯作者: 常天俊 E-mail:changtj@ hpu.edu.cn
  • 基金资助:
    国家自然科学基金(批准号：U1704241)和河南省科技攻关项目(批准号：242102321046)资助

Engineering Stimuli-responsive RNA-cleaving DNAzymes in Organic Cosolvents

HE Rongrong，YOU Xin，ZHENG Xing，YANG Menghan，CHANG Tianjun   

  1. Institute of Resources and Environment，Henan Polytechnic University
  • Received:2025-01-09 Revised:2025-02-16 Online First:2025-02-20 Published:2025-02-20
  • Contact: Tian-Jun CHANG E-mail:changtj@ hpu.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China (No. U1704241) and the Science and Technology Research Project of Henan Province, China (No. 242102321046)

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摘要： 引入新的识别元件对脱氧核酶(DNAzyme)进行工程化是拓展其应用范围的重要途径，但这依赖于对其可工程化位点的系统表征。为此，本文对前期发现的需要有机溶剂的RNA-cleaving DNAzyme (RCD,名为E3)进行工程化改造，在其不同区域发现了多个可工程化位点，并构建了多种的刺激响应性RCD。首先在可变区域将E3劈成两半，通过设计“DNA拉链”构建多组分酶(Multiple Component DNAzyme, MNAzyme)的策略，在可变区鉴别了6个可用于工程化的位点；接着在可变区成功设计了由单个外加的DNA序列激活的MNAzyme系统；进一步在E3的底物结合臂发现了另一个可用于设计MNAzyme的区域，并设计了一系列需两个DNA引发剂同时存在才能激活的三组分酶系统。所有MNAzyme系统在35% DMSO和30%乙醇中都有极高的选择性和极低的背景值，多种MNAzyme和引发剂共存体系中的反应信号接近单个引发剂产生信号的加和。本研究发现为在含有机溶剂的应用场景中构建响应性RCD探针提供了平台。

关键词: 脱氧核酶, 刺激响应性, 有机溶剂, 分子工程, 多组分酶

Abstract: Engineering deoxyribozyme (DNAzyme) by the introduction of new recognition elements is an important approach for expanding its applications, however, this depends on the systematic characterization of the engineerable sites in the DNAzyme. In this work, we investigated the engineerable sites of an organic solvent-needing RNA-cleaving DNAzyme (RCD, named E3) previously reported by our group. We first split E3 into two parts at its variable region, and added two DNA sequences on the 5’-end of one part and 3’-end of the other part to form duplex to active the DNAzyme. By construction of the zipper-like Multiple Component DNAzyme (MNAzyme), we were able to identify 6 splitting sites that could be utilized for DNAzyme engineering. Based on this discovery, a DNAzyme system that was activated by an external DNA initiator was constructed by splitting E3 at its variable region and introducing two oligonucleotides at the split sites for the binding of the initiator. Furthermore, we identified several splitting sites on the substrate-binding arm of E3 as an alternative region for the design of MNAzyme. Application in both regions, an intricate three component DNAzyme system was designed to be activated only in the presence of the two specific initiators. All the MNAzyme systems exhibited extremely high selectivity and very low background both in 35% DMSO and 30% ethanol. Moreover, in mixing of various MNAzyme systems and all the relative initiators, the cleavage products were almost the sum of that in mixing these MNAzymes in the presence of individual initiator. These results demonstrate that E3 is an excellent scaffold for constructing responsive RCD probes in applications in organic solvents-containing scenarios.

Key words: DNAzyme; Stimuli-responsive, Organic solvent, Molecular engineering; Multiple component DNAzyme

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