Hsiao Jeff C, Buryska Tomas, Kim Eunjung, Howes Philip D, deMello Andrew J
Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, 8093 Zürich, Switzerland.
Division of Bioengineering and Department of Bioengineering and Nano-Bioengineering, Incheon National University, Incheon 22012, Republic of Korea.
Nanoscale. 2021 Mar 12;13(9):4956-4970. doi: 10.1039/d0nr08668a.
Enzyme-nanoparticle interactions can give rise to a range of new phenomena, most notably significant enzymatic rate enhancement. Accordingly, the careful study and optimization of such systems is likely to give rise to advanced biosensing applications. Herein, we report a systematic study of the interactions between nuclease enzymes and oligonucleotide-coated gold nanoparticles (spherical nucleic acids, SNAs), with the aim of revealing phenomena worthy of evolution into functional nanosystems. Specifically, we study two nucleases, an exonuclease (ExoIII) and an endonuclease (Nt.BspQI), via fluorescence-based kinetic experiments, varying parameters including enzyme and substrate concentrations, and nanoparticle size and surface coverage in non-recycling and a recycling formats. We demonstrate the tuning of nuclease activity by SNA characteristics and show that the modular units of SNAs can be leveraged to either accelerate or suppress nuclease kinetics. Additionally, we observe that the enzymes are capable of cleaving restriction sites buried deep in the oligonucleotide surface layer and that enzymatic rate enhancement occurs in the target recycling format but not in the non-recycling format. Furthermore, we demonstrate a new SNA phenomenon, we term 'target stacking', whereby nucleic acid hybridization efficiency increases as enzyme cleavage proceeds during the beginning of a reaction. This investigation provides important data to guide the design of novel SNAs in biosensing and in vitro diagnostic applications.
酶与纳米颗粒的相互作用能够引发一系列新现象，其中最显著的是酶促反应速率大幅提高。因此，对这类系统进行细致研究和优化，有望催生先进的生物传感应用。在此，我们报告了一项关于核酸酶与寡核苷酸包覆的金纳米颗粒（球形核酸，SNA）之间相互作用的系统研究，旨在揭示那些值得发展为功能性纳米系统的现象。具体而言，我们通过基于荧光的动力学实验，研究了两种核酸酶，一种是外切核酸酶（ExoIII），另一种是内切核酸酶（Nt.BspQI），实验中改变了包括酶和底物浓度、纳米颗粒大小以及非循环和循环模式下的表面覆盖率等参数。我们证明了SNA特性可调节核酸酶活性，并表明SNA的模块化单元可用于加速或抑制核酸酶动力学。此外，我们观察到这些酶能够切割深埋在寡核苷酸表面层的限制性位点，并且酶促反应速率提高发生在目标循环模式而非非循环模式中。此外，我们展示了一种新的SNA现象，我们称之为“目标堆积”，即在反应开始时，随着酶切割的进行，核酸杂交效率会提高。这项研究提供了重要数据，以指导在生物传感和体外诊断应用中新型SNA的设计。
