Rose Galvan Angelica, Tiboni-Wyatt Isabella, Mathur Divita, Green Christopher M, He Ye, Ulijn Rein V, Medintz Igor L, Díaz Sebastián A
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, D.C. 20375, United States.
Fischell Department of Bioengineering, College of Engineering, University of Maryland, College Park, Maryland 20742, United States.
Biomacromolecules. 2025 Sep 8;26(9):5767-5777. doi: 10.1021/acs.biomac.5c00600. Epub 2025 Aug 13.
We demonstrate that coacervates, membraneless organelles formed by liquid-liquid phase separation, sequester and protect short DNA reporters and a functional luciferase gene from enzymatic degradation by various nucleases. Associative coacervates, formed by electrostatic interactions between polyhistidine peptides and ATP, inhibit degradation very efficiently. This protection arises from strong electrostatic interactions between the peptides and oligonucleotides, limiting the enzyme access to recognition and active sites. In contrast, simple coacervates based on a sticker-and-spacer model peptide exhibited limited protection. Oligonucleotide release from associative coacervates can be triggered by external stimuli such as ionic strength or temperature increases, enabling controlled release. Using a cell-free transcription-translation system, we demonstrated that in the presence of nucleases, the associative coacervate samples protected and maintained luciferase production. The ability to protect and controllably release functional genetic material makes coacervates promising candidates for further development as biocompatible delivery vehicles and components of cell-free synthetic biology platforms.
我们证明，凝聚体（通过液-液相分离形成的无膜细胞器）能够隔离并保护短DNA报告基因和功能性荧光素酶基因免受各种核酸酶的酶促降解。由多组氨酸肽与ATP之间的静电相互作用形成的缔合凝聚体能够非常有效地抑制降解。这种保护作用源于肽与寡核苷酸之间强烈的静电相互作用，限制了酶对识别位点和活性位点的接触。相比之下，基于粘贴-间隔模型肽的简单凝聚体的保护作用有限。缔合凝聚体中寡核苷酸的释放可由离子强度或温度升高之类的外部刺激触发，从而实现可控释放。利用无细胞转录-翻译系统，我们证明在存在核酸酶的情况下，缔合凝聚体样本能够保护并维持荧光素酶的产生。保护和可控释放功能性遗传物质的能力使凝聚体有望进一步发展成为生物相容性递送载体以及无细胞合成生物学平台的组件。
