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Development of New Approaches for Characterising DNA Origami-Based Nanostructures with Atomic Force Microscopy and Super-Resolution Microscopy

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Development of New Approaches for Characterising DNA Origami-Based Nanostructures with Atomic Force Microscopy and Super-Resolution Microscopy Development of new approaches for characterising DNA origami-based nanostructures with atomic force microscopy and super-resolution microscopy Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Bereich Mathematik und Naturwissenschaften der Technischen Universität Dresden von M.Sc. Franziska Elisabeth Fischer geboren am 16. Dezember 1987 in Dresden, Deutschland eingereicht am 09.08.2018 Gutachter Prof. Dr. rer. nat. et Ing. habil. Michael Mertig Prof. em. Dr. rer. nat. Manfred Stamm verteidigt am 09.01.2019 Die Dissertation wurde in der Zeit von Januar 2013 bis August 2018 an der Professur für Physikalische Chemie, Mess- und Sensortechnik und am cfaed - Center for Advancing Electronics Dresden, TU Dresden - angefertigt. For Mom. For Dad. “When morning comes, you would better find yourself saying: ’I have so many choices of what to do or what to leave - every morning, every day. I better judge for myself, and - go ahead and do it.’” Stefan Hell something Contents Abstract 3 1 Introduction 5 1.1 Objective and thesis outline . 5 1.2 DNA nanotechnology: structure assembly and current research areas . 8 1.2.1 Structure assembly . 8 1.2.2 Current fields of research . 14 1.3 Overview over the current techniques for the structural characterisation of DNA origami . 16 1.3.1 Gel electrophoresis . 17 1.3.2 Atomic force microscopy . 17 1.3.3 Fluorescence techniques . 19 1.3.4 Super-resolution microscopy . 20 1.4 Precision and trueness in SRM and multicolour co-localisation approaches . 23 1.5 Towards the assembly of CP molecules with DNA origami and possible applications . 26 1.5.1 Conjugated polymers . 26 1.5.2 Polythiophene and water-soluble, regioregular, end-functionalised derivatives via state-of-the-art synthesis . 29 1.5.3 Nanopatterning of conjugated polymers for single-molecule studies . 30 1.5.4 Nanopatterning of conjugated polymers with DNA . 32 1.5.5 State-of-the-art polymer-DNA origami hybrids . 33 2 Materials and Methods 37 2.1 Chemicals . 37 2.2 DNA origami synthesis . 37 2.3 AuNP and AuNR attachment to the pads and tPads . 38 2.4 Formation of P3(EO)3T-b-ODN BCPs and hybrids of BCPs and origami pads . 39 2.5 Methods used on multiple occasions . 40 2.6 Atomic force microscopy . 42 2.6.1 Sample preparation for scanning in air and high-resolution scanning in liquid . 42 2.6.2 Melting studies . 42 2.6.3 Locating of the BCP in hybrids in high-resolution liquid AFM images using the FindFoci algorithm . 42 2.7 Super-resolution fluorescence microscopy . 45 2.7.1 Protocols . 45 2.7.2 Error propagation . 49 3 Construction and characterisation of two 2D DNA origami structures with emphasis on AFM-based investigations 51 3.1 Design, synthesis and characterisation of pad and tPad . 51 1 3.1.1 Design and synthesis . 51 3.1.2 Structure evaluation . 53 3.1.3 Yield determination and synthesis variations to improve the yield . 56 3.1.4 Thermal and mechanical stability . 60 3.1.5 Stacking . 65 3.2 Towards decorating DNA origami . 66 3.2.1 Design of the link sequences . 66 3.2.2 Testing the binding capabilities of the links . 67 3.2.3 Testing the pegboard qualities of tPads and pads by arranging nanoscale gold . 68 3.3 Summary . 70 4 Qualitative and quantitative characterisation of polymer-DNA origami hybrids with AFM 73 4.1 Three step hybrid fabrication . 73 4.2 Results of the quantitative analysis of high-resolution AFM images of polyhiophene-DNA origami hybrids . 80 4.2.1 Object size . 83 4.2.2 Number of objects and object positions on the different pad types . 84 4.3 Influence of the scanning on the samples and limitations of AFM . 93 4.4 Summary and outlook . 95 5 Characterisation of surface-deposited 2D DNA origami with multicolour super-resolution microscopy 97 5.1 Basic principles, origami under test and method procedure . 97 5.1.1 Basic principles and labelling . 98 5.1.2 The two tPad designs . 99 5.1.3 Method procedure . 100 5.2 Results . 104 5.2.1 Colour offset after mapping . 105 5.2.2 Characterising the structural integrity of origami samples . 110 5.2.3 Reliability and robustness of the multicolour method . 124 5.3 Summary . 128 6 Summary and Outlook 131 6.1 Thesis summary . 131 6.2 Discussion and future tasks . 133 Appendix 137 A Additional images 139 B EGNAS 151 C DNA sequences 153 D List of abbreviations 165 List of Figures 199 List of Tables 201 2 Abstract DNA nanotechnology has developed a versatile set of methods to utilise DNA self-assembly for the bottom-up construction of arbitrary two- and three-dimensional DNA objects in the nanometre size range, and to functionalise the structures with unprecedented site-specificity with nanoscale objects such as metallic and semiconductor nanoparticles, proteins, fluorescent dyes, or synthetic polymers. The advances in structure assembly have resulted in the application of functional DNA-based nanostructures in a gamut of fields from nanoelectronic circuitry, nanophotonics, sensing, drug delivery, to the use as host structure or calibration standard for different types of microscopy. However, the analytical means for characterising DNA-based nanostructures drag behind these advances. Open questions remain, amongst others in quantitative single-structure evaluation. While techniques such as atomic force microscopy (AFM) or transmission electron microscopy (TEM) offer feature resolution in the range of few nanometres, the number of evaluated structures is often limited by the time-consuming manual data analysis. This thesis has introduced two new approaches to quantitative structure evaluation using AFM and super-resolution fluorescence microscopy (SRM). To obtain quantitative data, semi-automated computational image analysis routines were tailored in both approaches. AFM was used to quantify the attachment yield and placement accuracy of poly(3-tri(ethylene glycol)thiophene)-b-oligodeoxynucleotide diblock copolymers on a rectangular DNA origami. This work has also introduced the first hybrid of DNA origami and a conjugated polymer that uses a highly defined polythiophene derivative synthesised via state-of-the-art Kumada catalyst-transfer polycondensation. Among the AFM-based studies on polymer-origami-hybrids, this was the first to attempt near-single molecule resolution, and the first to introduce computational image analysis. Using the FindFoci tool of the software ImageJ revealed attachment yields per handle between 26 - 33 %, and determined a single block copolymer position with a precision of 80 - 90 %. The analysis has pointed out parameters that potentially influence the attachment yield such as the handle density and already attached objects. Furthermore, it has suggested interactions between the attached polymer molecules. The multicolour SRM approach used the principles of single-molecule high-resolution co-localisation (SHREC) to evaluate the structural integrity and the deposition side of the DNA origami frame “tPad” based on target distances and angles in a chiral fluorophore pattern the tPads were labelled with. The computatinal routine that was developed for image analysis utilised clustering to identify the patterns in a sample’s signals and to determine their characteristic distances and angles for hundreds of tPads simultaneously. The method excluded noise robustly, and depicted the moderate proportion of intact tPads in the samples correctly. With a registration error in the range of 10 -15 nm after mapping of the colour channels, the precision of a single distance measurements on the origami appeared in the range of 20 - 30 nm. By broadening the scope of computational AFM image analysis and taking on a new SRM approach for structure analysis, this work has presented working approaches towards new tools for quantitative analysis in DNA nanotechnology. Furthermore, the work has presented a new approach to constructing hybrid structures from DNA origami and conjugated polymers, which will open up new possibilities in the construction of nanoelectronic and nanophotonic structures. 3 4 Chapter 1 Introduction 1.1 Objective and thesis outline Entering the nanoscale in science and technology has shaped the world we live in today. It is accountable for the remarkable progress in information technology (with the 10 nm node currently being introduced[1]), has led to new kinds of functional materials in construction, clothing, or coatings and created new, interdisciplinary fields in physics, chemistry, medicine, and other sciences. Currently, nanofabrication relies on top-down methods. These methods, e. g., lithography, use large devices to carve smaller structures from bulk material, which reaches its limits as the structure sizes reach the molecular, or eventually the atomic level. Bottom-up processes build nanostructures from sub-nanometre precursors. Currently, they offer a strategy to augment the top-down methods, but hold the potential to replace it. Some bottom-up methods like dip-pen lithography, electron beam lithography or focused ion beam lithography use technically advanced, macroscopic instruments to manipulate individual molecules or atoms. Using controlled particle self-assemblies, however, requires less expensive equipment and promises a higher throughput. Outstanding self-assembled structures occur in nature. Biological molecules like proteins, RNA, carbohydrates, lipids and DNA form target structures with sub-nanometre precision, for example, protein capsules of viruses, motor proteins that conduct linear or rotary movements, or lipid membranes. The self-assembly of a DNA double strand from two single strands is the most understood of the aforementioned processes to date. The field of DNA nanotechnology has developed routines to build artificial nanosized structures from DNA, and hybrids of those and various other nanomaterials. For the formation of such DNA-based nanostructures, robust and efficient protocols have been established in the past 30 years. As a result of its maturing, the field is moving on to exploring functionality and the application of DNA-based nanostructures in a gamut of fields such as nanomedicine, fluorescence microscopy, molecular scaffolding in nuclear magnetic resonance (NMR) spectroscopy or protein cryogenic electron microscopy (cryo-EM), structural biophysics, biosensing or plasmonics and photonics.
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