In dieser Arbeit werden systematisch die bisher unerforschten grundlegenden physikalischen und chemischen Eigenschaften von DNA-Origami untersucht, die die Stabilität dieser aus doppelsträngiger DNA aufgebauten nanoskopischen Suprastrukturen bestimmen. In Analogie zu den zahlreichen Studien, die sich mit der Stabilität von Proteinen durch kontrollierte Denaturierung beschäftigen, spielen auch in dieser Arbeit die Denaturierungsbedingungen eine zentrale Rolle. Unter Verwendung von Guanidinium (Gdm+) als teilweise DNA-stabilisierendes, aber auch potentiell denaturierendes Kation steht dessen Wirkung auf DNA-Origami-Dreiecke im Mittelpunkt der Untersuchungen, wobei insbesondere die unerwartete Modulation der nanoskopischen Schädigung von DNA-Origami durch die begleitenden Gegenanionen zu Gdm+ im Vordergrund steht. Die Experimente zielen darauf ab, atomistische, molekulare, nanoskopische und thermodynamische Eigenschaften von DNA-Origami zu korrelieren und zu klären, wie diese vom Design des DNA-Origami selbst abhängen können.
Die Ergebnisse zeigen einen unerwarteten Zusammenhang zwischen den spezifischen Gegenanionen des Denaturierungsmittels und der Stabilität der DNA-Origami-Dreiecke: Sulfat wirkt stabilisierend, während Chlorid die Superstruktur bereits unterhalb der globalen Schmelztemperatur destabilisiert. Statistische Analysen von Rasterkraftmikroskop (AFM)-Bildern und Zirkulardichroismus (CD)-Spektren zeigen Strukturübergänge auf nano-skopischer bzw. molekularer Ebene. Werden diese Techniken mit thermischer Denaturierung in Gegenwart von schwacher bis starker chemischer Denaturierung kombiniert, so zeigt sich, dass Änderungen der Wärmekapazität (ΔCp) während der strukturellen Veränderungen der DNA-Originale eine Schlüsselrolle bei der Bestimmung ihrer Empfindlichkeit gegenüber Temperatur und Denaturierungsmitteln spielen. Die Daten deuten darauf hin, dass Wasser auf apolaren DNA-Origami-Oberflächen der molekulare Ursprung der abgeleiteten Wärme-kapazitätsänderungen ist. Diese Hypothese wird durch Molekulardynamik-Simulationen (MD) unterstützt, die die Modulation von ΔCp durch die Hydratationshüllen der Anionen zeigen. Ihr unterschiedliches Potential, stabile Ionenpaare mit Gdm+ in konzentrierten Salzlösungen zu bilden, kann die experimentell beobachteten Variationen der strukturellen Stabilität erklären. Die Kopplung von strukturellen Übergängen an ΔCp wird somit als Schlüsselfaktor für die Destabilisierung von DNA-Origami sowohl bei höheren als auch bei niedrigeren Temperaturen identifiziert. Darüber hinaus weisen DNA-Origami nicht nur diese Eigenschaft auf, sondern ermöglichen auch die Beobachtung von kalten Denaturierungsprozessen auf nanoskopischer Ebene, bei denen kälteinduzierte Spannungen innerhalb der Superstruktur bei einem Bruch an vorherbestimmten lokalen Stellen freigesetzt werden, die in AFM-Bildern sichtbar sind. Dies ist die erste Beobachtung der kälteinduzierten Denaturierung von Nukleinsäuren bei Temperaturen über 0 °C sowie von DNA-basierten Superstrukturen.
In dieser Arbeit wird die strukturelle Stabilität von sechs verschiedenen 2D- und 3D-DNA-Origami-Nanostrukturen in unterschiedlichen chemischen Umgebungen untersucht. Drei chaotrope Salze - Guanidiniumsulfat (Gdm2SO4), Guanidiniumchlorid (GdmCl) und Tetrapropylammoniumchlorid (TPACl) - werden als Denaturierungsmittel verwendet. Mittels Rasterkraftmikroskopie wird die Integrität der Nanostrukturen quantifiziert, wobei sich Gdm2SO4 als das schwächste und TPACl als das stärkste Denaturierungsmittel für DNA-Origami erweist, was sich auch in den Schmelztemperaturen widerspiegelt. Die Abhängigkeit der DNA-Origami-Stabilität von der Superstruktur wird besonders bei 3D-Nanostrukturen deutlich. Hier zeigen mechanisch flexible Designs sowohl in GdmCl als auch in TPACl eine höhere Stabilität als ihre starren Gegenstücke. Die Abhängigkeit der DNA-Origami-Stabilität von der Superstruktur wird besonders in 3D-Nanostrukturen deutlich, in denen mechanisch flexible Strukturen sowohl in GdmCl als auch in TPACl eine höhere Stabilität aufweisen als ihre steifen Gegenstücke. Dies begünstigt die Bildung von intramolekularen Verformungen, die sich entweder in 'weichen' Architekturen über die gesamte Superstruktur verteilen oder in ansonsten 'steifen' Strukturen in den weniger stabilen Regionen konzentrieren.:Table of contents
Questions addressed in this thesis .................................................................................... I
Abstract ................................................................................................................................ I
Englisch .................................................................................................................................................. I
Deutsch .................................................................................................................................................. II
Acronyms ........................................................................................................................... III
Substances .......................................................................................................................................... IV
Physical and Chemical abbreviations ............................................................................................... IV
Mathematical abbreviations ................................................................................................................ V
1 Introduction ................................................................................................................. 1
1.1
Deoxyribonucleic acid ................................................................................................................. 1
1.1.1 The structure of DNA ............................................................................................................ 1
1.1.2 Hydrogen bonds .................................................................................................................... 1
1.1.3 Base stacking ........................................................................................................................ 2
1.1.4 Water DNA interactions: A complex dance of stability and dynamics .................................. 5
1.1.5 The effect of ionic strength on DNA conformation ................................................................ 9
1.1.6 Conformational changes ....................................................................................................... 9
1.1.7 Forms of DNA ..................................................................................................................... 10
1.1.8 The role of apolar groups in DNA unfolding ........................................................................ 13
1.1.9 Energetics of DNA structural transitions ............................................................................. 14
1.1.10 Melting temperature ............................................................................................................ 15
1.2
Hofmeister series ....................................................................................................................... 17
1.2.1 Probing the Hofmeister series: Salt effects biomolecules ................................................... 17
1.2.2 Specific ion effects in electrolyte solutions .......................................................................... 19
1.3
DNA nanostructures................................................................................................................... 20
1.3.1 DNA origami ........................................................................................................................ 22
1.3.2 Challenges in DNA origami stability .................................................................................... 26
1.3.3 DNA origami in single molecule studies .............................................................................. 27
1.4
Circular dichroism ...................................................................................................................... 28
1.4.1 Circular dichroism spectroscopy for analyzing DNA conformations ................................... 30
1.4.2 Wavelength-dependent spectroscopic signatures of DNA conformation ........................... 32
1.5
Atomic force microscopy .......................................................................................................... 34
1.6
2D correlation spectroscopy ..................................................................................................... 36
1.6.1 2D correlation spectroscopy: Synchronous and asynchronous spectra analysis ............... 39
1.6.2 Perturbation-correlation moving-window 2D correlation spectroscopy ............................... 40
1.7
Multivariate analysis of spectral data using PCA and ITTFA ................................................ 41
1.8
Cold denaturation ....................................................................................................................... 42
2 Results and Discussion ............................................................................................ 44
2.1
Cold denaturation of the Rothemund DNA origami triangle .................................................. 45
2.2
Heat denaturation of the Rothemund DNA origami triangle .................................................. 50
2.2.1 Investigations by atomic force microscopy ......................................................................... 51
2.2.2 Circular dichroism spectroscopy and thermodynamic modelling ........................................ 56
2.2.3 Divergent effects of Cl- and SO42- on DNA origami stability ................................................ 62
2.3
Magnesium concentration modulation of DNA Origami heat denaturation ......................... 65
2.4
Assessing DNA origami stability in different chaotropic environments .............................. 66
2.4.1 DNA origami integrity influenced by Gdm2SO4 ................................................................... 67
2.4.2 DNA origami integrity influenced by GdmCl ........................................................................ 73
2.4.3 DNA origami integrity influenced by TPACl ........................................................................ 75
2.4.4 Quantitative comparison ..................................................................................................... 77
3 Critics ......................................................................................................................... 78
4 Conclusion ................................................................................................................. 79
5 Outlook ...................................................................................................................... 81
6 Material and Methods ................................................................................................ 82
6.1
DNA origami synthesis .............................................................................................................. 82
6.2
Sample preparation and AFM imaging ..................................................................................... 82
6.2.1 Anion-specific structure and stability of guanidinium-bound DNA origami & Cold denaturation of DNA origami nanostructures ...................................................................... 82
6.2.2 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 83
6.2.3 Cold denaturation of DNA origami nanostructures ............................................................. 83
6.3
CD spectroscopy and analysis ................................................................................................. 84
6.3.1 Anion-specific structure and stability of guanidinium-bound DNA origami ......................... 84
6.3.2 Pre-treatment of the CD data and calculation of melting temperatures .............................. 84
6.3.3 Cold denaturation of DNA origami nanostructures ............................................................. 84
6.3.4 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 84
6.4
Principal component analysis and iterative target test factor analysis ............................... 85
6.5
Thermodynamic modelling ........................................................................................................ 85
6.6
Molecular dynamics modelling ................................................................................................. 85
Appendix ........................................................................................................................... 88
Acknowledgment ............................................................................................................ 100
Bibliography .................................................................................................................... 101
List of Figures ................................................................................................................. 116
List of Tables ................................................................................................................... 118
Declaration of independence – Selbstständigkeitserklärung ...................................... 119 / This thesis undertakes the systematic study of hitherto unexplored fundamental physical and chemical properties of DNA origami that determine the stability of these designed nanoscopic superstructural assemblies of double-stranded DNA. In analogy to the vast number of studies addressing protein stability by controlled denaturation, denaturing conditions play a central role in this thesis as well. Using guanidinium (Gdm+) as a partly DNA-stabilizing but also potentially denaturing cation, its effect on DNA origami triangles is central to the study which particularly addressed the unexpected modulation of nanoscopic damage of DNA origami by the accompanying counter-anions to Gdm+. The experiments aim at correlating atomistic, molecular, nanoscopic and thermodynamic properties of DNA origami and at elucidating how these may depend on the DNA origami design itself.
The results demonstrate an unexpected relationship between the specific counter-anions of the denaturant and the stability of DNA origami triangles: sulphate exhibits stabilizing effects and chloride induces destabilization of the superstructure already below the global melting temperature. Statistical analyses of both atomic force microscopy (AFM) images and circular dichroism (CD) spectra reveal structural transitions at the nanoscopic and molecular level, respectively. Combining these techniques with thermal denaturation in the presence of mild to strong chemical denaturation, changes in heat capacity (ΔCp) during DNA origami structural changes are shown to play the key role in determining their sensitivity to temperature and denaturants. The data suggest that water at apolar DNA origami surfaces is the molecular origin of the derived heat capacity changes. This hypothesis is substantiated by Molecular Dynamics (MD) simulations which shed light on the modulation of ΔCp by the hydration shells of anions. Their different potential to form stable ion pairs with Gdm+ in concentrated salt solutions can explain the experimentally observed variations of structural stability.
The coupling of structural transitions to ΔCp is thus identified as a key factor in the destabilization of DNA origami at both elevated and lowered temperatures. Furthermore, DNA origami not only exhibit this property, but also enable the observation of cold denaturation processes at the nanoscopic level, where cold-induced strain within the superstructure is released upon breakage at predisposed local sites, visible in AFM images. This is the first observation of cold-induced denaturation of nucleic acids at temperatures above 0 °C, as well of DNA-based superstructures.
Extending the scope, the work evaluates the structural stability of six different 2D and 3D DNA origami nanostructures in different chemical environments. Three chaotropic salts - guanidinium sulfate (Gdm2SO4), guanidinium chloride (GdmCl), and tetrapropylammonium chloride (TPACl) - are used as denaturants. Atomic force microscopy quantifies the nanostructural integrity, revealing Gdm2SO4 as the weakest and TPACl as the strongest DNA origami denaturant, which is also reflected in the melting temperatures. The dependence of DNA origami stability on its superstructure is particularly evident in 3D nanostructures, where mechanically flexible designs exhibit higher stability in both GdmCl and TPACl than rigid counterparts. This supports the buildup of intramolecular strain, which becomes either partitioned among the entire superstructure in “soft” architectures or accumulates at the least stable regions in otherwise “rigid” designs.:Table of contents
Questions addressed in this thesis .................................................................................... I
Abstract ................................................................................................................................ I
Englisch .................................................................................................................................................. I
Deutsch .................................................................................................................................................. II
Acronyms ........................................................................................................................... III
Substances .......................................................................................................................................... IV
Physical and Chemical abbreviations ............................................................................................... IV
Mathematical abbreviations ................................................................................................................ V
1 Introduction ................................................................................................................. 1
1.1
Deoxyribonucleic acid ................................................................................................................. 1
1.1.1 The structure of DNA ............................................................................................................ 1
1.1.2 Hydrogen bonds .................................................................................................................... 1
1.1.3 Base stacking ........................................................................................................................ 2
1.1.4 Water DNA interactions: A complex dance of stability and dynamics .................................. 5
1.1.5 The effect of ionic strength on DNA conformation ................................................................ 9
1.1.6 Conformational changes ....................................................................................................... 9
1.1.7 Forms of DNA ..................................................................................................................... 10
1.1.8 The role of apolar groups in DNA unfolding ........................................................................ 13
1.1.9 Energetics of DNA structural transitions ............................................................................. 14
1.1.10 Melting temperature ............................................................................................................ 15
1.2
Hofmeister series ....................................................................................................................... 17
1.2.1 Probing the Hofmeister series: Salt effects biomolecules ................................................... 17
1.2.2 Specific ion effects in electrolyte solutions .......................................................................... 19
1.3
DNA nanostructures................................................................................................................... 20
1.3.1 DNA origami ........................................................................................................................ 22
1.3.2 Challenges in DNA origami stability .................................................................................... 26
1.3.3 DNA origami in single molecule studies .............................................................................. 27
1.4
Circular dichroism ...................................................................................................................... 28
1.4.1 Circular dichroism spectroscopy for analyzing DNA conformations ................................... 30
1.4.2 Wavelength-dependent spectroscopic signatures of DNA conformation ........................... 32
1.5
Atomic force microscopy .......................................................................................................... 34
1.6
2D correlation spectroscopy ..................................................................................................... 36
1.6.1 2D correlation spectroscopy: Synchronous and asynchronous spectra analysis ............... 39
1.6.2 Perturbation-correlation moving-window 2D correlation spectroscopy ............................... 40
1.7
Multivariate analysis of spectral data using PCA and ITTFA ................................................ 41
1.8
Cold denaturation ....................................................................................................................... 42
2 Results and Discussion ............................................................................................ 44
2.1
Cold denaturation of the Rothemund DNA origami triangle .................................................. 45
2.2
Heat denaturation of the Rothemund DNA origami triangle .................................................. 50
2.2.1 Investigations by atomic force microscopy ......................................................................... 51
2.2.2 Circular dichroism spectroscopy and thermodynamic modelling ........................................ 56
2.2.3 Divergent effects of Cl- and SO42- on DNA origami stability ................................................ 62
2.3
Magnesium concentration modulation of DNA Origami heat denaturation ......................... 65
2.4
Assessing DNA origami stability in different chaotropic environments .............................. 66
2.4.1 DNA origami integrity influenced by Gdm2SO4 ................................................................... 67
2.4.2 DNA origami integrity influenced by GdmCl ........................................................................ 73
2.4.3 DNA origami integrity influenced by TPACl ........................................................................ 75
2.4.4 Quantitative comparison ..................................................................................................... 77
3 Critics ......................................................................................................................... 78
4 Conclusion ................................................................................................................. 79
5 Outlook ...................................................................................................................... 81
6 Material and Methods ................................................................................................ 82
6.1
DNA origami synthesis .............................................................................................................. 82
6.2
Sample preparation and AFM imaging ..................................................................................... 82
6.2.1 Anion-specific structure and stability of guanidinium-bound DNA origami & Cold denaturation of DNA origami nanostructures ...................................................................... 82
6.2.2 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 83
6.2.3 Cold denaturation of DNA origami nanostructures ............................................................. 83
6.3
CD spectroscopy and analysis ................................................................................................. 84
6.3.1 Anion-specific structure and stability of guanidinium-bound DNA origami ......................... 84
6.3.2 Pre-treatment of the CD data and calculation of melting temperatures .............................. 84
6.3.3 Cold denaturation of DNA origami nanostructures ............................................................. 84
6.3.4 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 84
6.4
Principal component analysis and iterative target test factor analysis ............................... 85
6.5
Thermodynamic modelling ........................................................................................................ 85
6.6
Molecular dynamics modelling ................................................................................................. 85
Appendix ........................................................................................................................... 88
Acknowledgment ............................................................................................................ 100
Bibliography .................................................................................................................... 101
List of Figures ................................................................................................................. 116
List of Tables ................................................................................................................... 118
Declaration of independence – Selbstständigkeitserklärung ...................................... 119
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:92614 |
| Date | 01 August 2024 |
| Creators | Dornbusch, Daniel |
| Contributors | Fahmy, Karim, Diez, Stefan, Hof, Martin, Technische Universität Dresden, Helmholtz-Zentrum Dresden-Rossendorf e.V. (HZDR) |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | German |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 10.1016/j.csbj.2022.05.037, 10.1039/d3nr02045b, 10.1039/D3CC05985E |
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