Center for Molecular Design and Biomimetics at the Biodesign Institute and School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, United States.
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States.
ACS Synth Biol. 2021 Nov 19;10(11):3066-3073. doi: 10.1021/acssynbio.1c00336. Epub 2021 Nov 9.
In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.
在核酸纳米技术中，链置换是一种广泛应用的机制，其中一条杂交双链体的链被入侵链取代，入侵链与双链体上的一个结合点（单链区域）结合。它被用于在分子水平上执行逻辑运算、引发级联反应，甚至用于诊断和治疗。虽然已经进行了系统的实验研究来探测具有不同结合点长度、序列和错配位置的 DNA 中链置换的动力学，但对 RNA 或 RNA-DNA 杂交系统还没有进行类似的研究。在这里，我们通过实验研究了结合点长度、结合点位置（链的 5' 端或 3' 端）和错配如何影响链置换动力学。我们观察到随着结合点长度的增加和结合点位于底物的 5' 端，反应加速。我们发现，与离界面更远的错配相比，更接近结合点和双链体界面的错配会使反应减慢。RNA 与 DNA 置换与杂交置换（RNA 入侵 DNA 或 DNA 入侵 RNA）的比较部分可以用各自结合点区域的热力学稳定性来解释，但也表明在 RNA 入侵 DNA 的情况下，从 B 型螺旋到 A 型螺旋的重排可能在动力学中起作用。
