Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures
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 choleste...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-11-01
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| Series: | Membranes |
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| Online Access: | https://www.mdpi.com/2077-0375/11/12/950 |
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| author | Jasleen Kaur Daljit Singh Minh Tri Luu Jonathan F. Berengut Ali Abbas Matthew A. B. Baker Shelley F. J. Wickham |
| author_facet | Jasleen Kaur Daljit Singh Minh Tri Luu Jonathan F. Berengut Ali Abbas Matthew A. B. Baker Shelley F. J. Wickham |
| author_sort | Jasleen Kaur Daljit Singh |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T03:36:00Z |
| format | Article |
| id | doaj.art-42b7d137ed634ec2b84a50cb53a9c524 |
| institution | Directory Open Access Journal |
| issn | 2077-0375 |
| language | English |
| last_indexed | 2024-03-10T03:36:00Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Membranes |
| spelling | doaj.art-42b7d137ed634ec2b84a50cb53a9c5242023-11-23T09:30:21ZengMDPI AGMembranes2077-03752021-11-01111295010.3390/membranes11120950Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami NanostructuresJasleen Kaur Daljit Singh0Minh Tri Luu1Jonathan F. Berengut2Ali Abbas3Matthew A. B. Baker4Shelley F. J. Wickham5School of Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSchool of Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSchool of Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSchool of Chemical and Biomolecular Engineering, University of Sydney, Sydney, NSW 2006, AustraliaSchool of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, AustraliaSchool of Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaDNA 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.https://www.mdpi.com/2077-0375/11/12/950DNA origamiDNA nanotechnologycholesterolaggregation |
| spellingShingle | Jasleen Kaur Daljit Singh Minh Tri Luu Jonathan F. Berengut Ali Abbas Matthew A. B. Baker Shelley F. J. Wickham Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures Membranes DNA origami DNA nanotechnology cholesterol aggregation |
| title | Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures |
| title_full | Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures |
| title_fullStr | Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures |
| title_full_unstemmed | Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures |
| title_short | Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures |
| title_sort | minimizing cholesterol induced aggregation of membrane interacting dna origami nanostructures |
| topic | DNA origami DNA nanotechnology cholesterol aggregation |
| url | https://www.mdpi.com/2077-0375/11/12/950 |
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