DNA origami nanostructures as molecular rulers to measure membrane thickness
Lead Research Organisation:
King's College London
Department Name: Randall Div of Cell and Molecular Biophy
Abstract
Lipids, or fat molecules, are very important parts of cells. Cells produce thousands of different lipids and hundreds of proteins to make and transport these lipids, but it is largely unknown why cells invest so much energy into maintaining such a diverse population of lipids. The goal of this interdisciplinary project is to understand how lipids, and especially their side-chains, participate in important biological events. The student will make and characterise a DNA nanostructure-based molecular ruler. This will, for the first time, result in a tool to measure membrane thickness in live cells.
Organisations
People |
ORCID iD |
| Ulrike Eggert (Primary Supervisor) | |
| Sioned Jones (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
| 1902642 | Studentship | BB/M009513/1 | 01/10/2017 | 02/03/2022 | Sioned Jones |
| Description | DNA nanostructures can be equipped with hydrophobic anchors to promote interaction with lipid bilayer membranes. However, the factors that govern interaction is not well understood, and most studies are limited to synthetic membranes. To rationally design a DNA-based molecular ruler, we investigated the design features that influence DNA-membrane interaction. We have conducted a systematic study on the anchoring efficiency of hydrophobic DNA duplexes to membranes of synthetic vesicles and live cells. We designed duplexes with a broad range of hydrophobic anchors, including cholesterol lipid anchors and charge-neutral alkyl-phosphorothioate belts differing in alkyl chain length and position. The duplexes were synthesised and characterised using gel electrophoresis and mass spectrometry. A fluorescent Cy3-tag was incorporated into each duplex to allow microscopic detection. The interaction of the duplexes with lipid bilayers of GFP-encapsulated giant unilamellar vesicles (GUVs) was studied. The cholesterol-modified duplex anchored to the lipid bilayer membrane of GUVs of varied composition and charge. Ethyl-PPT duplexes did not bind to any GUVs, while longer chain and more hydrophobic hexyl-PPT bound only to neutrally-charged GUVs. The PPT belt position also influenced membrane interaction, with the terminal belt more favourable for membrane association than the centre. Analysis of the relative membrane fluorescence intensities using Fiji software revealed enhanced fluorescence with the cholesterol anchor compared to the hexyl-PPT belt. A Cy3-labelled duplex with no hydrophobic modifications was used as a control throughout this study and did not interact with membranes of GUVs. We investigated the interaction of the hydrophobic duplexes in biological bilayers. We incubated the duplexes with live MyrPalm-GFP HeLa cells and imaged using the spinning-disc confocal microscope. The cholesterol duplex anchored to the plasma membrane and co-localised to the Myrpalm-GFP fluorescence. Both ethyl and hexyl-PPT duplexes also associated with the plasma membrane, ethyl-PPT displayed vesicular fluorescence while hexyl-PPT assembled to higher-order structures at the plasma membrane. Information on orientation and mode of binding (tethering vs membrane-spanning) was obtained for the various anchors using nuclease digestion assays. The results of this study show that DNA nanostructures can be rationally designed to facilitate specific and varied membrane interaction. This study will aid the rational design of DNA-based molecular rulers to achieve insertion and spanning into lipid bilayers of live cells and measure the membrane thickness. |
| Exploitation Route | Our study is the first to provide comparative data on the relation between hydrophobic anchors and lipid bilayer membranes to help the rational design of DNA nanostructures for more specific and suitable applications. We anticipate our study a starting point for designing membrane-interacting DNA nanostructures, and subsequently, help advance their use in biophysical applications. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |