Daljit Singh Jasleen Kaur, Luu Minh Tri, Berengut Jonathan F, Abbas Ali, Baker Matthew A B, Wickham Shelley F J
School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia.
School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, NSW 2006, Australia.
Membranes (Basel). 2021 Nov 30;11(12):950. doi: 10.3390/membranes11120950.
DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with diverse functions, such as shaping membranes, tethering defined numbers of membrane proteins, and transmembrane nanopores. The modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and the inhibition of membrane interaction. Here, we quantify the assembly yield of two cholesterol-modified DNA origami nanostructures: a 2D DNA origami tile (DOT) and a 3D DNA origami barrel (DOB), by gel electrophoresis. We found that the DOT assembly yield (relative to the no cholesterol control) could be maximised by reducing the number of cholesterols from 6 to 1 (2 ± 0.2% to 100 ± 2%), optimising the separation between adjacent cholesterols (64 ± 26% to 78 ± 30%), decreasing spacer length (38 ± 20% to 95 ± 5%), and using protective ssDNA 10T overhangs (38 ± 20% to 87 ± 6%). Two-step folding protocols for the DOB, where cholesterol strands are added in a second step, did not improve the yield. Detergent improved the yield of distal cholesterol configurations (26 ± 22% to 92 ± 12%), but samples re-aggregated after detergent removal (74 ± 3%). Finally, we confirmed functional membrane binding of the cholesterol-modified nanostructures. These findings provide fundamental guidelines to reducing the cholesterol-induced aggregation of membrane-interacting 2D and 3D DNA origami nanostructures, improving the yield of well-formed structures to facilitate future applications in nanomedicine and biophysics.
DNA纳米技术提供了构建具有多种功能的定制膜相互作用纳米结构的方法，例如塑造膜、连接特定数量的膜蛋白以及跨膜纳米孔。为促进膜相互作用，需要用疏水性基团（如胆固醇）对DNA纳米结构进行修饰。然而，胆固醇诱导的DNA折纸纳米结构聚集仍然是一个挑战。聚集会导致组装产率降低、结构缺陷以及膜相互作用受到抑制。在此，我们通过凝胶电泳对两种胆固醇修饰的DNA折纸纳米结构：二维DNA折纸片（DOT）和三维DNA折纸桶（DOB）的组装产率进行了量化。我们发现，通过将胆固醇数量从6个减少到1个（从2±0.2%提高到100±2%）、优化相邻胆固醇之间的间距（从64±26%提高到78±30%）、缩短间隔长度（从38±20%提高到95±5%）以及使用保护性单链DNA 10T突出端（从38±20%提高到87±6%），DOT的组装产率（相对于无胆固醇对照）可以最大化。DOB的两步折叠方案（即在第二步添加胆固醇链）并没有提高产率。去污剂提高了远端胆固醇构型的产率（从26±22%提高到92±12%），但去除去污剂后样品会重新聚集（74±3%）。最后，我们证实了胆固醇修饰的纳米结构与膜的功能性结合。这些发现为减少胆固醇诱导的膜相互作用二维和三维DNA折纸纳米结构聚集提供了基本指导方针，提高了结构良好的结构的产率，以促进未来在纳米医学和生物物理学中的应用。
