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DNAzymes as Activity-Based Sensors for Metal Ions: Recent Applications, Demonstrated Advantages, Current Challenges, and Future Directions.
Accounts of Chemical Research ( IF 18.3 ) Pub Date : 2019-11-13 , DOI: 10.1021/acs.accounts.9b00419
Ryan J. Lake , Zhenglin Yang , JingJing Zhang , Yi Lu

Metal ions can be beneficial or toxic depending on their identity, oxidation state, and concentration. Therefore, the ability to detect and quantify different types of metal ions using portable sensors or in situ imaging agents is important for better environmental monitoring, in vitro medical diagnostics, and imaging of biological systems. While numerous metal ions in different oxidation states are present in the environment and biological systems, only a limited number of them can be detected effectively using current methods. In this Account, we summarize research results from our group that overcome this limitation by the development of a novel class of activity-based sensors based on metal-dependent DNAzymes, which are DNA molecules with enzymatic activity. First, we have developed an in vitro selection method to obtain DNAzymes from a large DNA library of up to 1015 sequences that can carry out cleavage of an oligonucleotide substrate only in the presence of a specific metal ion with high selectivity. Negative selection steps can further be used to improve the selectivity against potentially competing targets by removing sequences that recognize the competing metal ions. Second, we have developed a patented catalytic beacon method to transform the metal-dependent DNAzyme cleavage reaction into a turn-on fluorescent signal by attaching a fluorophore and quenchers to the DNAzyme complex. Because of the difference in the melting temperatures of DNA hybridization before and after metal-ion-dependent cleavage of the DNAzyme substrate, the fluorophore on the DNA cleavage product can be released from its quenchers to create a turn-on fluorescent signal. Because DNAzymes are easy to conjugate with other signaling moieties, such as gold nanoparticles, lanthanide-doped upconversion nanoparticles, electrochemical agents, and gadolinium complexes, these DNAzymes can also readily be converted into colorimetric sensors, upconversion luminescence sensors, electrochemical sensors, or magnetic resonance contrast agents. In addition to describing recent progress in developing and applying these metal ion sensors for environmental monitoring, point-of-care diagnostics, cellular imaging, and in vivo imaging in zebrafish, we summarize major advantages of this class of activity-based sensors. In addition to advantages common to most activity-based sensors, such as enzymatic turnovers that allow for signal amplification and the use of initial rates instead of absolute signals for quantification to avoid interferences from sample matrices, the DNAzyme-based sensors allow for in vitro selection to expand the method to almost any metal ion under a variety of conditions, negative selection to improve the selectivity against competing targets, and reselection of DNAzymes and combination of active and inactive variants to fine-tune the dynamic range of detection. The use of melting temperature differences to separate target binding from signaling moieties in the catalytic beacon method allows the use of different fluorophores and nanomaterials to extend the versatility and modularity of this sensing platform. Furthermore, sensing and imaging artifacts can be minimized by using an inactive mutant DNAzyme as a negative control, while spatiotemporal control of sensing/imaging can be achieved using optical, photothermal, and endogenous orthogonal caging methods. Finally, current challenges, opportunities, and future perspectives for DNAzymes as activity-based sensors are also discussed.

中文翻译:

DNA酶作为金属离子的基于活性的传感器:最近的应用,已证明的优势,当前的挑战和未来的方向。

金属离子可能是有益的还是有毒的,具体取决于它们的身份,氧化态和浓度。因此,使用便携式传感器或原位成像剂检测和定量不同类型金属离子的能力对于更好的环境监测,体外医学诊断和生物系统成像很重要。尽管环境和生物系统中存在许多处于不同氧化态的金属离子,但是使用当前的方法只能有效地检测到有限数量的金属离子。在此报告中,我们总结了我们小组的研究结果,这些研究结果通过开发基于金属依赖性DNA酶的新型一类基于活动的传感器来克服了这一局限,该酶是具有酶活性的DNA分子。第一的,我们已经开发了一种体外选择方法,可从多达1015个序列的大型DNA库中获得DNA酶,仅在存在特定金属离子的情况下,才能以高选择性进行寡核苷酸底物的裂解。阴性选择步骤可以进一步用于通过去除识别竞争性金属离子的序列来提高针对潜在竞争性靶标的选择性。其次,我们开发了一种专利的催化信标方法,通过将荧光团和淬灭剂连接到DNAzyme络合物上,将依赖金属的DNAzyme裂解反应转变为开启的荧光信号。由于DNA酶底物的金属离子依赖性切割前后DNA杂交的解链温度不同,DNA裂解产物上的荧光团可以从其淬灭剂中释放出来,从而产生开启的荧光信号。因为DNA酶很容易与其他信号传导部分偶联,例如金纳米颗粒,掺杂镧系元素的上转换纳米颗粒,电化学试剂和g络合物,所以这些DNA酶也可以很容易地转换为比色传感器,上转换发光传感器,电化学传感器或磁共振造影剂。除了描述开发和应用这些金属离子传感器用于斑马鱼的环境监测,即时诊断,细胞成像和体内成像的最新进展外,我们还总结了此类基于活动的传感器的主要优点。除了大多数基于活动的传感器共有的优势外,例如允许信号放大的酶促转换以及使用初始速率代替绝对信号进行定量分析以避免来自样品基质的干扰,基于DNAzyme的传感器允许进行体外选择,从而将方法扩展到几乎所有金属离子。条件,阴性选择以提高对竞争靶标的选择性,以及DNA酶的重新选择以及有活性和无活性变体的组合以微调检测的动态范围。在催化信标方法中使用解链温度差将靶标结合与信号传导部分分开,可以使用不同的荧光团和纳米材料来扩展该传感平台的多功能性和模块化。此外,通过使用无活性的突变型DNAzyme作为阴性对照,可以最大程度地减少感测和成像伪影,而可以使用光学,光热和内源正交笼化方法实现对感测/成像的时空控制。最后,还讨论了DNA酶作为基于活动的传感器的当前挑战,机遇和未来展望。
更新日期:2019-11-14
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