Responsive Hydrogels for Label-Free Signal Transduction within Biosensors

Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications

Harnessing the physicochemical properties of DNA as a multifunctional biomaterial for biomedical and other applications

The biological purpose of DNA is to store, replicate, and convey genetic information in cells. Progress in molecular genetics have led to its widespread applications in gene editing, gene therapy, and forensic science. However, in addition to its role as a genetic material, DNA has also emerged as a nongenetic, generic material for diverse biomedical applications. DNA is essentially a natural biopolymer that can be precisely programed by simple chemical modifications to construct materials with desired mechanical, biological, and structural properties. This review critically deciphers the chemical tools and strategies that are currently being employed to harness the nongenetic functions of DNA. Here, the primary product of interest has been crosslinked, hydrated polymers, or hydrogels. State-of-the-art applications of macroscopic, DNA-based hydrogels in the fields of environment, electrochemistry, biologics delivery, and regenerative therapy have been extensively reviewed. Additionally, the review encompasses the status of DNA as a clinically and commercially viable material and provides insight into future possibilities

DNA nanostructures for biotechnological applications

Deoxyribonucleic acid (DNA) is a versatile biomolecule which can be used for the rational design and assembly of nanoscale structures. This thesis explores the use of functional DNA- enzyme nanostructures for applications in biocatalysis and for directed motion on the nanoscale. In the first part of this thesis, a DNA scaffold was outlined for the display and immobilization of enzyme cascades. Confinement or spatial organization of enzyme cascades is adopted in biological systems to prevent loss of reactive intermediates and to facilitate substrate conversion in chemically complex and crowded intracellular environments. We adopt a strategy to create concentrated enzyme assemblies directed by a DNA structure generated by F29 rolling circle amplification (RCA). These DNA assemblies, DNA nanoballs, were investigated for the display of two bi-enzyme systems. Firstly, a horseradish peroxidase and glucose oxidase enzyme pair, and secondly, a transaminase and norcoclaurine synthase bi- enzyme system for the synthesis of biotechnologically relevant benzylisoquinoline (BIA) precursors. The second part of this thesis concerns the use of enzymatic catalysis as a means of affecting the motion of a nanoscale DNA structure. Molecular movement on the micro and nanoscales is a fundamental feature of biological systems, and recreating this functionality represents an important step in the realization of intelligent synthetic devices for directed transport and chemotaxis in response to stimuli. While directed motion has been shown for DNA structures on predefined tracks to which they are hybridized, enzymatic catalysis has not been investigated as an approach to controlling the motion of DNA nanostructures. We show that the motion of a DNA structure tethered to multiple lysine decarboxylase molecules is enhanced by its substrate, L-lysine the ‘fuel’, in a concentration dependent manner, based on nanoparticle tracking analysis (NTA) and DLS analyses

Engineered Materials to Measure and Regulate Cell Mechanotransduction

The extracellular environment plays a key role in a wide array of cellular functions including migration, tissue formation, and differentiation. This thesis overviews the design of a molecular sensor to measure cellular forces and a hydrogel system to engineer angiogenic sprouting. We developed molecular force probes (FPs) that report traction forces of adherent cells with high spatial resolution, can be linked to virtually any surface, and do not require monitoring deformations of elastic substrates. FPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific amounts of force. In chapter two we overview the synthetic strategies to produce these FPs from solid-state synthesis. We then demonstrate the chemical and physical characterization of these FPs. These data show that the FPs can be designed rationally from existing knowledge of the force-responsiveness of DNA hairpins. Chapter three summarizes our methods to affix these FPs to solid substrates to measure cellular traction forces. The silane chemistry to conjugate these FPs to glass coverslips is reported in detail. Then, the results of converting the fluorescence of these FPs to force values is given along with biological validation. We find using this method that cellular tractions are exerted at the distal ends of focal adhesions. In chapter four we present a versatile bioactive PEG hydrogel to study angiogenesis. This material is MMP-degradable and cell-adhesive. We show a microfabrication strategy to micromold these gels to pattern angiogenic sprouting from ex vivo tissue explants

RecA-templated DNA scaffolds for selective site-specific assembly of nanoparticles for electronic devices

With today’s challenges in the electronic industry, novel alternative ap- proaches for manufacturing devices at nanoscale are being investigated. Using self-assembly, arguably has the best potential for nanostructures. DNA and proteins - some of the most important biomolecules use self- assembly extensively for natural functions. Chemical and structural pre- dictability of DNA and specificity of proteins promise a big potential for novel materials and could allow creation of structures controlled at nanoscale level. RecombinaseA - a DNA-binding protein has been used for controllable and predictable patterning of selected DNA sequences, opening the way to nanometre-scale DNA marking. However, protein patterning alone does not add any electric or other desired functionality to the DNA, there- fore additional modifications are neccessary. Furthermore, since biologi- cal molecules have transient functionality, system stability investigation is crucial for needed modification and subsequent usage. This project focused on RecA-patterned DNA modification for electric prop- erty addition. Thiolation and subsequent attachment of gold or magnetic nanoparticles to RecA protein present on DNA were investigated as a method for creating electrically conductive nanoscale objects. More specifically, at- tachment of gold nanoparticles throughout the whole patterned region of DNA and attachment of single nanoparticles at precise positions were looked into. The work successfully demonstrated that both nanoparticle deposition along the full length of RecA-coated DNA and specific single nanoparticle positioning is feasible. For investigating RecA-DNA stability, a system based on FRET was de- vised and used to analyse interaction kinetics. It was found that RecA-DNA complexes are fully formed in minutes and stay bound for hours. Specific configurations of the set-up showed distinct lack of signal, suggesting com- plicated interactions between the protein and patterned DNA. The project demonstrated through binding of NPs at specific locations and on the whole filament length that the system has potential for electronic applications and its stability is sufficient for processing times

Optymalizacja procesów transferu energii i transferu elektronowego w biofotowoltaicznych nanourządzeniach zawierających fotosystem I oraz cytochrom c553 z ekstremofilnego krasnorostu Cyanidioschyzon merolae

One of the biggest challenges of modern-day solar technologies is to develop carbon-neutral, efficient and sustainable systems for solar energy conversion into electricity and fuel. Over the last two decades there has been a growing impact of ‘green’ solar conversion technologies based on the natural solar energy converters, such as the robust extremophilic photosystem I (PSI) and its associated protein cofactors. The main bottleneck of the currently available biophotovoltaic and solar-to-fuel technologies is the low power conversion efficiency of the available devices due to wasteful charge recombination reactions at the interfaces between the working modules, as well as instability of the organic and inorganic components. This thesis describes the development of three novel approaches to improve energy and electron transfer in PSI-based biophotoelectrodes and plasmonic nanostructures: (1) construction of all-solid-state mediatorless biophotovoltaic devices incorporating p-doped silicon substrate, extremophilic robust PSI complex and its associated light harvesting antenna (PSI-LHCI) in conjunction with its natural electron donor cytochrome c553 (cyt c553) from a red microalga Cyanidioschyzon merolae and (2), biofunctionalization of the silver nanowires (AgNWs) with a highly organised architecture of the cyt c553/PSI-LHCI assembly for the significant improvement of absorption cross-section of the C. merolae PSI-LHCI complex due to plasmonic interactions between the distinct subpool of chlorophylls (Chls) and AgNWs nanoconstructs. The third (3) approach was based on development of the photo-driven in vitro hydrogen production system following hybridisation of the robust extremophilic PSI-LHCI complex with the novel and established proton reducing catalysts (PRC). The last approach has led to generation of molecular hydrogen with TOF of 521 mol H2 (mol PSI)-1 min-1 and 729 mol H2 (mol PSI)-1 min-1 for the hybrid systems of PSI-LHCI with cobaloxime and the DuBois-type mononuclear nickel proton reduction catalysts, respectively. The TOF values for biophotocatalytic H2 production obtained in this study were 3-fold and 16.6-fold higher than those published for cyanobacterial PSI/PRC hybrid systems employing cobaloxime and a similar Ni mononuclear PRC, respectively. Construction of all-solid-state mediatorless PSI-based nanodevices was facilitated by biopassivation of the p-doped Si substrate with His6-tagged cyt c553, as evidenced by significant lowering of the inherent dark saturation current (J0), a well-known semiconductor surface recombination parameter. Five distinct variants of cyt c553 were obtained by genetically engineering the specific linker peptides of 0-19 amino acids in length between the cyt c553 holoprotein and a C-terminal His6-tag, the latter being the affinity ‘anchor’ used for specific immobilisation of this protein on the semiconductor surface. The calculated 2D Gibbs free energy maps for all the five cyt c553 variants and the protein lacking any peptide linker showed a much higher number of thermodynamically feasible conformations for the cyt c variants containing longer linker peptides upon their specific immobilisation on the Si surface. The bioinformatic calculations were verified by constructing the respective cyt c553/Si bioelectrodes and measuring their dark current-voltage (J-V) characteristics to determine the degree of p-doped Si surface passivation, measured by minimisation of the J0 recombination parameter. The combined bioinformatic and J-V analyses indicated that the cyt c553 variants with longer linker peptides, up to 19AA in length, allowed for more structural flexibility of immobilised cyt c553 in terms of both, orientation and distance of the haem group with respect to the Si surface, resulting in efficient biopassivation of this semiconductor substrate. This molecular approach has allowed for the developing of an alternative, cheap and facile route for significant reduction of the inherent minority charge recombination at the p-doped Si surface. To improve direct electron transfer within all-solid state PSI-based nanodevices, the specific His6-tagged cyt c553 variants, generated in this study, were attached to the Ni-NTA-functionalised p-doped Si surface prior to incorporation of the PSI-LHCI photoactive layer. Such nanoarchitecture resulted in an open-circuit potential increment of 333 μV for the specific PSI-LHCI/cyt c553/Si nanodevice compared to the control device devoid of cyt c553. Moreover, the all-solid state mediatorless PSI-LHCI-based devices produced photocurrents in the range of 104-234 μA/cm2 when a bias of -0.25 V was applied, demonstrating one of the highest photocurrents for this type of solid-state devices reported to date. The power conversion efficiency of the PSI-LHCI/p-doped Si devices was 20-fold higher when 19AA variant of cyt c553 was incorporated as the biological conductive interface between the PSI-LHCI photoactive module and the substrate, demonstrating the significant role of this cyt variant for improving direct electron transfer within the PSI-based all-solid-state mediatorless biophotovoltaic device. In a complementary line of research, it was demonstrated that the highly controlled assembly of C. merolae PSI-LHCI complex on plasmon-generating AgNWs substantially improved the optical functionality of such a novel biohybrid nanostructure. By comparing fluorescence intensities measured for PSI-LHCI complex randomly oriented on AgNWs and the results obtained for the PSI-LHCI/cyt c553 bioconjugate with AgNWs it was concluded that the specific binding of PSI-LHCI complex with the defined uniform orientation yields selective excitation of a pool of Chls that are otherwise almost non-absorbing. This is remarkable, as this work shows for the first time that plasmonic excitations in metallic nanostructures not only can be used to enhance native absorption of photosynthetic pigments, but also, by employing cyt c553 as the conjugation cofactor, to activate the specific Chl pools as the absorbing sites, only when the uniform and well-defined orientation of PSI-LHCI complex with respect to plasmonic nanostructures is achieved. This innovative approach paves the way for the next generation solar energy-converting technologies to outperform the reported-to-date biohybrid devices with respect to power conversion efficiency.Jednym z głównych wyzwań technologicznych jest opracowanie wydajnych i odnawialnych systemów konwersji energii słonecznej w elektryczność i paliwo, stosując zerowy bilans emisji związków węgla. W ciągu ostatnich dwóch dekad nastąpił znaczący postęp w zastosowaniu “zielonych” technologii biofotowoltaicznych, opartych na naturalnych białkach absorbujących energię słoneczną, takich jak fotosystem I (PSI) wraz ze związanymi z nim kompleksami antenowymi i kofaktorami transportu elektronowego. Głównym ograniczeniem obecnych urządzeń fotowoltaicznych jest ich niska wydajność kwantowa, związana z procesami rekombinacji ładunku w interfejsach pomiędzy modułami tych urządzeń, jak również ograniczona stabilność zastosowanych jak dotąd biologicznych i syntetycznych komponentów. W ramach niniejszej rozprawy doktorskiej opracowano nowatorską technologię, polegającą na zastosowaniu wysokostabilnego PSI oraz naturalnego donora elektronów dla tego kompleksu, cytochromu c553 (cyt c553), wyizolowanych z ekstremofilnego krasnorostu Cyanidioschyzon merolae, do konstrukcji trzech typów nanourządzeń biofotowoltaicznych: (1), biofotoogniw w stałej konfiguracji (ang., all-solid-state), zawierających domieszkowany pozytywnie półprzewodnikowy substrat krzemowy (ang., p-doped Si, p-Si) wraz z warstwami fotoaktywnego kompleksu PSI i cyt c553; (2), plazmonowych srebrnych bionanodrutów (AgNWs), funkcjonalizowanych wysokouporządkowaną nanoarchitekturą monowarstw PSI i cyt c553, oraz (3), systemu fotokatalitycznej produkcji wodoru cząsteczkowego in vitro z zastosowaniem kompleksów hybrydowych PSI wraz z syntetycznymi katalizatorami redukcji protonów (ang., proton reducing catalysts, PRC). W przypadku ostatniego z powyższych systemów, optymalizacja biofotokatalitycznej produkcji wodoru cząsteczkowego z zastosowaniem systemów hybrydowych z PSI i PRC, opartych na kobaloksymie i niklowym katalizatorze mononuklearnym typu DuBois, precypitowanych na powierzchni PSI w roztworze wodnym, pozwoliła na osiągnięcie aktywności wydzielania wodoru odpowiednio, 521 moli H2 (mol PSI)-1 min-1 oraz 729 moli H2 (mol PSI)-1 min-1, przewyższając tym samym 3-17-krotnie aktywność wydzielania wodoru w podobnych systemach biohybrydowych i warunkach pomiarowych. Poraz pierwszy zastosowano cyt c553 z C-terminalną metką His6 do biopasywacji półprzewodnikowego substratu p-Si, mierzonej minimalizacją parametru rekombinacji powierzchniowej J0. Poprzez inżynierię genetyczną sklonowano i wyrażono w E. coli 5 różnych wariantów cyt c553, z których 4 zawierały w swej strukturze sekwencje peptydowe o długości 5-19 aminokwasów (AA), aby zbadać ich wpływ na procesy rekombinacji ładunku w obrębie elektrody krzemowej. Peptydy te zostały wstawione pomiędzy holobiałkiem a metką His6, którą zastosowano do unieruchomienia każdego z wariantów cyt c553 na powierzchni elektrody. Obliczenie energii swobodnej Gibbsa pozwoliło na utworzenie konformacyjnych map 2D dla każdego z wariantów, w których pokazano, iż warianty z semi-helikalnym peptydem 19AA przyjmują znacząco większą liczbę termodynamicznie możliwych konformacji na powierzchni elektrody pod względem odległości i kąta nachylenia grupy hemowej w stosunku do powierzchni elektrody. Bioinformatyczna analiza została potwierdzona poprzez ciemniową charakterystykę prądowo-napięciową (J-V) utworzonych odpowiednio bioelektrod krzemowo-cytochromowych. Stwierdzono, że warianty cyt c553 z dłuższymi peptydami pomiędzy metką His6 a holobiałkiem efektywnie minimalizują prądy ciemniowe krzemowego substratu, najprawdopodobniej dzięki istnieniu większej ilości termodynamicznie zoptymalizowanych konformacji cytochromu, pozwalających na minimalizację rekombinacji ładunku powierzchniowego substratu. Funkcjonalizacja elektrody p-Si wariantem cyt c553, charakteryzującym się największym stopniem swobody orientacji grupy hemowej w stosunku powierzchni elektrody krzemowej, pozwoliła na efektywną biopasywację tego półprzewodnikowego substratu poprzez minimalizację parametru J0, co z kolei pozwoliło na zwiększenie parametru Voc o 333 μV w biofotoogniwach typu PSI/cyt c553/p-Si, w porównaniu do kontroli zawierającej jedynie PSI/p-Si. Uzyskano fotoprądy w stałych biofotoogniwach PSI/p-Si w zakresie 104-234 μA cm-2 (przy nadpotencjale -0.25 V), co należy do jednych z najwyższych wartości fotoprądów wygenerowanych przez stałe biofotoogniwa z PSI, w podobnych warunkach pomiarowych. Jednocześnie wydajność konwersji energii słonecznej w fotoogniwach typu PSI-LHCI/cyt c553/p-Si była 20-krotnie wyższa, w obecności wariantu cyt c553 19AA, zastosowanego w tych urządzeniech jako biologiczna warstwa biopasywacji substratu krzemowego oraz warstwa kondukcyjna pomiędzy substratem a PSI. Tym samym wykazano, że ów wariant może być zastosowany w urządzeniach biofotowoltaicznych do zwiększenia transferu elektronowego pomiędzy substratem a PSI. W równoległym i komplementarnym kierunku badań, zastosowanie równomiernej i specyficznie ukierunkowanej nanoarchitektury fotoaktywnej warstwy PSI na plazmonowych nanostrukturach metalicznych AgNWs, sfunkcjonalizowanych uprzednio cyt c553, pozwoliło na znaczące zwiększenie efektywnej absorpcji PSI, w zakresie spektralnym, w którym PSI jest nieaktywny in vivo, poprzez aktywację specyficznej puli tzw. czerwonych cząsteczek chlorofilu w obrębie fluoroforów PSI. Tym samym pokazano, że oddziaływania plazmonowe mogą być efektywnie zastosowane nie tylko do zwiększenia całkowitej absorpcji fotoaktywnych kompleksów białkowych, ale również do aktywacji spektralnej specyficznych pigmentów, wyłącznie w obrębie wysokouporządkowanej i zorientowanej nanoarchitektury tych fotokompleksów na nanokonstruktach plazmonowych. Powyższe nowatorskie podejście badawcze może być w przyszłości zastosowane do konstrukcji nowej generacji urządzeń biofotowoltaicznych o zwiększonej wydajności konwersji energii słonecznej

Spatial regulation of membrane receptor signaling using DNA origami

Juxtracrine signaling between apposing membrane receptors and ligands is an important class of intercellular communication. Much focus has been directed towards studying the biochemical interactions between receptors and ligands, their surface expression levels and signaling activities for driving downstream signaling processes. However, the lateral distribution of receptors/ligands on the membrane has been gaining increasing significance in modulating intercellular signaling. Nevertheless, little is known about the cellular mechanisms of interpreting this biophysical factor during ligand/receptor signaling. The work in thesis is based on the hypothesis that cells use information from the spatial organization of their surface ligands/receptors to direct intracellular signaling. To address this, we have employed the power of DNA origami technology to manipulate ligand spatial distances with nanometer precision and constrain their cognate receptors into defined configurations in ephrin/Eph signaling and the T-cell negative regulators PD-L1/PD-1 on T cell signaling. With this approach, we demonstrated that modulating the nanoscale organization of ephrin-A5 ligands contributed to divergent transcriptional profiles in human glioblastoma cells (paper I). We also showed that the nanoscale organization of PD-L1 regulates T-cell activation and sizes of PD-1 clusters (paper II). In summary, this work describes that the spatial organization of ligands/receptors at the nanoscale can serve as an important physical guidance cue that tunes the overall cellular response

Development of Diverse Size and Shape RNA Nanoparticles and Investigation of their Physicochemical Properties for Optimized Drug Delivery

RNA nanotechnology is an emerging field that holds great promise for advancing drug delivery and materials science. Recently, RNA nanoparticles have seen increased use as an in vivo delivery system. RNA was once thought to have little potential for in vivo use due to biological and thermodynamic stability issues. However, these issues have been solved by: (1) Finding of a thermodynamically stable three-way junction (3WJ) motif; (2) Chemical modifications to RNA confer enzymatic stability in vivo; and (3) the finding that RNA nanoparticles exhibit low immunogenicity in vivo. In vivo biodistribution and pharmacokinetics are affected by the physicochemical properties, such as size, shape, stability, and surface chemistry/properties, of the nanoparticles being delivered. RNA has an inherent advantage for nanoparticle construction as each of these properties can be finely tuned. The focus of this study is as follows: (1) Construction of diverse size and shape RNA nanoparticles with tunable physicochemical properties; (2) Investigation of the effect that size, shape, and nanoparticle properties have on in vivo biodistribution; (3) Development of drug encapsulation and release mechanism utilizing RNA nanotechnology; and (4) Establishment of large-scale synthesis and purification methods of RNA nanoparticles. In (1), RNA triangle, square, and pentagon shaped nanoparticles were constructed using the phi29 pRNA-3WJ as a core motif. Square nanoparticles were constructed with sizes of 5, 10, and 20 nanometers. The RNA polygons were characterized by AFM to demonstrate formation of their predicted geometry per molecular models. Furthermore, the properties of RNA polygons were tuned both thermodynamically and chemically by substitution of nucleic acid type used during nanoparticle assembly. In (2), the biodistribution of RNA nanosquares of diverse sizes and RNA polygons of diverse shapes were investigated using tumor models in nude mice. It was found that increasing the size of the nanosquares led to prolonged circulation time in vivo and higher apparent accumulation in the tumor. However, it was observed that changing of shape had little effect on biodistribution. Furthermore, the effect of the hydrophobicity on RNA nanoparticles biodistribution was examined in mouse models. It was found that incorporation of hydrophobic ligands into RNA nanoparticles causes non-specific accumulation in healthy organs, while incorporation of hydrophilic ligands does not. Lower accumulation in vital organs of hydrophobic chemicals was observed after conjugation to RNA nanoparticles, suggesting RNA has the property to solubilize hydrophobic chemicals and reduce accumulation and toxicity in vital organs. In (3), a 3D RNA nanoprism was constructed to encapsulate a small molecule fluorophore acting as a model drug. The fluorophore was held inside the nanoprism by binding to an RNA aptamer. The ability of the stable frame of the nanoprism to protect the fragile aptamer inside was evidenced by a doubling of the fluorescent half-life in a degrading environment. In (4), a method for large-scale in vitro synthesis and purification of RNA nanoparticles was devised using rolling circle transcription (RCT). A novel method for preparing circular double stranded DNA was developed, overcoming current challenges in the RCT procedure. RCT produced more than 5 times more RNA nanoparticles than traditional run-off transcription, as monitored by gel electrophoresis and fluorescence monitoring. Finally, large-scale purification methods using rate-zonal and equilibrium density gradient ultracentrifugation, as well as gel electrophoresis column, were developed

Nucleic Acid Architectures for Therapeutics, Diagnostics, Devices and Materials

Nucleic acids (RNA and DNA) and their chemical analogs have been utilized as building materials due to their biocompatibility and programmability. RNA, which naturally possesses a wide range of different functions, is now being widely investigated for its role as a responsive biomaterial which dynamically reacts to changes in the surrounding environment. It is now evident that artificially designed self-assembling RNAs, that can form programmable nanoparticles and supra-assemblies, will play an increasingly important part in a diverse range of applications, such as macromolecular therapies, drug delivery systems, biosensing, tissue engineering, programmable scaffolds for material organization, logic gates, and soft actuators, to name but a few. The current exciting Special Issue comprises research highlights, short communications, research articles, and reviews that all bring together the leading scientists who are exploring a wide range of the fundamental properties of RNA and DNA nanoassemblies suitable for biomedical applications

DNA-Based Mimics of Membrane Proteins Lipid-DNA Interactions Determine Function

Nucleic acids, particularly DNA, are used as a nanoscale building material, due to their unique controllability via complementarity of base pairing. One of the potential applications of DNA nanotechnology is creating synthetic constructs mimicking function of membrane proteins. These natural molecular machines function embedded in the lipid bilayer. Similar membrane attachment of DNA-based structures is achieved by modifying the nucleic acid with hydrophobic anchors, most commonly cholesterol. Aiming at developing a fully functional and controllable synthetic membrane construct, the first step I undertook was to understand and utilize fundamental interactions between molecules: DNA, cholesterol and lipids. Instead of starting with a complicated DNA-based model mimicking protein architecture, here I have created a set of simple systems that allowed me to examine the major interactions between involved molecules. This work describes four aspects of the DNA-lipid systems that I have built and studied experimentally. Firstly, I have analysed the effects of membrane-spanning DNA duplex on the lipids’ arrangement in the pore and presented how this arrangement can be remodelled depending on the hydrophilicity of the DNA design. Secondly, I have looked at the same system from the opposite perspective - studied and prevented the distortion of the transmembrane DNA construct induced by the surrounding lipids. Thirdly, I have evaluated the importance of ions in mediating DNA-lipid interactions, reporting analysis of two electrostatic phenomena: screening and bridging. Finally, utilizing a nanoengineered four-helix structure, I discussed surfactant’s influence on DNA membrane insertion efficiency, showing that aggregation of the nanostructures is one of the major factors determining their spontaneous membrane-spanning. While the understanding of phenomena in minimalistic systems is crucial for further development of complex pore-forming constructs, here I showed that even simple DNA nanostructures, when rationally designed, can mimic functionality of natural membrane proteins.EPSRC; Winton Programme for the Physics of Sustainabilit