Real time monitoring of DNA hybridization and replication using optical and acoustic biosensors

Während in den letzten Jahren zahlreiche Biosensoren zum spezifischen Nachweis von DNA entwickelt wurden, ist die Anwendung oberflächen-sensitiver Methoden auf enzymatische Reaktionen ein vergleichsweise neues Forschungsgebiet. Trotz der hohen Empfindlichkeit und der Möglichkeit zur Echtzeit-Beobachtung molekularer Prozesse, ist die Anwendung dieser Methoden nicht etabliert, da die Enzymaktivität durch die Nähe zur Oberfläche beeinträchtigt sein kann. Im Rahmen dieser Arbeit wurde die enzymatische Verlängerung immobilisierter DNA durch eine DNA Polymerase mit Hilfe von Oberflächenplasmonen-Fluoreszenzspektroskopie (SPFS) und einer Quarzkristall-Mikrowaage (QCM) untersucht. Die Synthese von DNA wurde im Fall der QCM als Massenzuwachs detektiert, der sich im Abfall der Resonanzfrequenz des Schwingquarzes und einem Anstieg seiner Dissipationsenergie ausdrückte. Die viskoelastischen Eigenschaften der DNA-Schichten wurden bestimmt, indem die erhaltenen Daten mit einem auf Voigt basierenden Modell ausgewertet wurden. SPFS nutzt das evaneszente elektromagnetische Feld, das mit Oberflächenplasmonen einhergeht, zur oberflächen-sensitiven Anregung von Chromophoren. Auf diese Weise wurde der Einbau von Farbstoff-markierten Nukleotiden in die entstehende DNA-Sequenz als Indikator für das Voranschreiten der Reaktion ausgenutzt. Beide Meßtechniken konnten erfolgreich zum Nachweis der DNA-Synthese herangezogen werden, wobei die katalytische Aktivität des Enzyms vergleichbar zu der in Lösung gemessenen war.

Investigation of the electric field enhanced PNA-DNA hybridization on Au surface by using surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

Recently, the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was developed as a kinetic analysis and a detection method with dual- monitoring of the change of reflectivity and fluorescence signal for the interfacial phenomenon. A fundamental study of PNA and DNA interaction at the surface using surface plasmon fluorescence spectroscopy (SPFS) will be investigated in studies. Furthermore, several specific conditions to influence on PNA/DNA hybridization and affinity efficiency by monitoring reflective index changes and fluorescence variation at the same time will be considered. In order to identify the affinity degree of PNA/DNA hybridizaiton at the surface, the association constant (kon) and the dissociation constant (koff) will be obtained by titration experiment of various concentration of target DNA and kinetic investigation. In addition, for more enhancing the hybridization efficiency of PNA/DNA, a study of polarized electric field enhancement system will be introduced and performed in detail. DNA is well-known polyelectrolytes with naturally negative charged molecules in its structure. With polarized electrical treatment, applying DC field to the metal surface, which PNA probe would be immobilized at, negatively charged DNA molecules can be attracted by electromagnetic attraction force and manipulated to the close the surface area, and have more possibility to hybridize with probe PNA molecules by hydrogen bonding each corresponding base sequence. There are several major factors can be influenced on the hybridization efficiency.

Kinetic and affinity analysis of hybridization reactions between PNA probes and DNA targets using surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

Sequenz spezifische biomolekulare Analyseverfahren erweisen sich gerade im Hinblick auf das Humane Genom Projekt als äußerst nützlich in der Detektion von einzelnen Nukleotid Polymorphismen (SNPs) und zur Identifizierung von Genen. Auf Grund der hohen Anzahl von Basenpaaren, die zu analysieren sind, werden sensitive und effiziente Rastermethoden benötigt, welche dazu fähig sind, DNA-Proben in einer geeigneten Art und Weise zu bearbeiten. Die meisten Detektionsarten berücksichtigen die Interaktion einer verankerten Probe und des korrespondierenden Targets mit den Oberflächen. Die Analyse des kinetischen Verhaltens der Oligonukleotide auf der Sensoroberfläche ist infolgedessen von höchster Wichtigkeit für die Verbesserung bereits bekannter Detektions - Schemata. In letzter Zeit wurde die Oberflächen Plasmonen feld-verstärkte Fluoreszenz Spektroskopie (SPFS) entwickelt. Sie stellt eine kinetische Analyse - und Detektions - Methode dar, die mit doppelter Aufzeichnung, d.h. der Änderung der Reflektivität und des Fluoreszenzsignals, für das Interphasen Phänomen operiert. Durch die Verwendung von SPFS können Kinetikmessungen für die Hybridisierung zwischen Peptid Nukleinsäure (PNA), welche eine synthetisierte Nukleinsäure DNA imitiert und eine stabilere Doppelhelix formt, und DNA auf der Sensoroberfläche ausgeführt werden. Mittels einzel-, umfassend-, und titrations- Experimenten sowohl mit einer komplementär zusammenpassenden Sequenz als auch einer mismatch Sequenz können basierend auf dem Langmuir Modell die Geschwindigkeitskonstanten für die Bindungsreaktion des oligomer DNA Targets bzw. des PCR Targets zur PNA ermittelt werden. Darüber hinaus wurden die Einflüsse der Ionenstärke und der Temperatur für die PNA/DNA Hybridisierung in einer kinetischen Analyse aufgezeigt.

DNA block copolymers – from micelles to machines

DNA block copolymer, a new class of hybrid material composed of a synthetic polymer and an oligodeoxynucleotide segment, owns unique properties which can not be achieved by only one of the two polymers. Among amphiphilic DNA block copolymers, DNA-b-polypropylene oxide (PPO) was chosen as a model system, because PPO is biocompatible and has a Tg < 0 °C. Both properties might be essential for future applications in living systems. During my PhD study, I focused on the properties and the structures of DNA-b-PPO molecules. First, DNA-b-PPO micelles were studied by scanning force microscopy (SFM) and fluorescence correlation spectroscopy (FCS). In order to control the size of micelles without re-synthesis, micelles were incubated with template-independent DNA polymerase TdT and deoxynucleotide triphosphates in reaction buffer solution. By carrying out ex-situ experiments, the growth of micelles was visualized by imaging in liquid with AFM. Complementary measurements with FCS and polyacrylamide gel electrophoresis (PAGE) confirmed the increase in size. Furthermore, the growing process was studied with AFM in-situ at 37 °C. Hereby the growth of individual micelles could be observed. In contrast to ex-situ reactions, the growth of micelles adsorbed on mica surface for in-situ experiments terminated about one hour after the reaction was initiated. Two reasons were identified for the termination: (i) block of catalytic sites by interaction with the substrate and (ii) reduced exchange of molecules between micelles and the liquid environment. In addition, a geometrical model for AFM imaging was developed which allowed deriving the average number of mononucleotides added to DNA-b-PPO molecules in dependence on the enzymatic reaction time (chapter 3). Second, a prototype of a macroscopic DNA machine made of DNA-b-PPO was investigated. As DNA-b-PPO molecules were amphiphilic, they could form a monolayer at the air-water interface. Using a Langmuir film balance, the energy released owing to DNA hybridization was converted into macroscopic movements of the barriers in the Langmuir trough. A specially adapted Langmuir trough was build to exchange the subphase without changing the water level significantly. Upon exchanging the subphase with complementary DNA containing buffer solution, an increase of lateral pressure was observed which could be attributed to hybridization of single stranded DNA-b-PPO. The pressure versus area/molecule isotherms were recorded before and after hybridization. I also carried out a series of control experiments, in order to identify the best conditions of realizing a DNA machine with DNA-b-PPO. To relate the lateral pressure with molecular structures, Langmuir Blodgett (LB) films were transferred to highly ordered pyrolytic graphite (HOPG) and mica substrates at different pressures. These films were then investigated with AFM (chapter 4). At last, this thesis includes studies of DNA and DNA block copolymer assemblies with AFM, which were performed in cooperation with different group of the Sonderforschungsbereich 625 “From Single Molecules to Nanoscopically Structured Materials”. AFM was proven to be an important method to confirm the formation of multiblock copolymers and DNA networks (chapter 5).

Struktur, Funktion und potentielle Anwendung der PKC- und SNZ,SNO-Promotoren aus Geodia cydonium und Suberites domuncula

Die Schwämme (Porifera) sind eine reiche Quelle bioaktiver Naturstoffe. Viele dieser Naturstoffe besitzen das Potential, als Pharmazeutika, molekulare Sonden usw. eingesetzt oder weiterentwickelt zu werden. Die Beschaffung dieser Naturstoffe in ausreichenden Mengen stellt jedoch eines der größten Probleme bei der Testung und Produktion vielversprechender Substanzen dar. Der Transfer von DNA in Schwammzellen bzw. in komplette Organismen wäre ein vielversprechender Ansatz, dieses Problem zu lösen. Das Ziel dieser Arbeit war es deshalb, die Funktion und Struktur homologer Promotoren zu untersuchen und eine Methode des Gentransfers in Schwammzellen auszuarbeiten. Zu diesem Zweck wurde zusätzlich zu der bereits vorhandenen 5'-flankierenden Region des conventional PKC-Gens aus Geodia cydonium eine genomische Bibliothek von Suberites domuncula konstruiert, um diese mit Hilfe des DNA-Homologiescreenings nach den 5'-flankierenden Regionen des cPKC- und des SNZ (SnooZe)-Gens (SD_SNZG) zu durchsuchen. Die Klonierung und Sequenzierung sowohl des 5'-Bereichs als auch die Charakterisierung der Exon-Intron Struktur beider Gene wurde erfolgreich durchgeführt. In der 5'-Region des SNZ-Gens konnte dabei ein weiteres Gen (SD_SNO; SNZ proximal Open Reading Frame) identifiziert werden, das in einer 'Kopf-an-Kopf' Anordnung zu SD_SNZG orientiert ist. Sowohl SD_SNZG als auch SD_SNO wurden hochkonservierten Genfamilien zugeordnet, deren Vorkommen in Metazoen hier erstmals beschrieben wird.Funktionelle Studien mit Hilfe der Reportergene Luciferase und Enhanced Green Fluorescent Protein (EGFP) im heterologen System der NIH 3T3 Zellen wiesen sowohl dem cPKC-Promotor aus G. cydonium als auch dem SNZ-Promotor aus S. domuncula eine starke Promotoraktivität im Verhältnis zum SV40-Promotor nach. Die Aktivität des cPKC-Promotors aus S. domuncula dagegen war relativ schwach. Darüber hinaus konnte geklärt werden, daß die 5'-flankierende Region des SNZ-Gens bidirektionale Promotoraktivität aufweist und daß der G. cydonium cPKC-Promotor keine TATA-Box besitzt, sondern eine GC-Box für die basale Funktion benötigt.Als geeignete Methode zur Transfektion von Zellen des Schwamms S. domuncula erwies sich der ballistische Gentransfer mit Hilfe der Gene Gun. Homologe Promotoren konnten die sichtbare Expression des Reportergens EGFP jedoch nicht bewirken. Nur der virale CMV-Promotor erwies sich als hierfür geeignet.

Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy

The research interest of this study is to investigate surface immobilization strategies for proteins and other biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique. The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with other functional molecules make this protein a prime candidate for supramolecular architectures. The recrystallization behavior on gold or on the secondary cell wall polymer (SCWP) was recorded by SPR. The optical thicknesses and surface densities for different protein layers were calculated. In DNA hybridization tests performed in order to discriminate different mismatches, recombinant S-layer-streptavidin fusion protein matrices showed their potential for new microarrays. Moreover, SCWPs coated gold chips, covered with a controlled and oriented assembly of S-layer fusion proteins, represent an even more sensitive fluorescence testing platform. Additionally, S-layer fusion proteins as the matrix for LHCII immobilization strongly demonstrate superiority over routine approaches, proving the possibility of utilizing them as a new strategy for biomolecular coupling. In the study of the SPFS hCG immunoassay, the biophysical and immunological characteristics of this glycoprotein hormone were presented first. After the investigation of the effect of the biotin thiol dilution on the coupling efficiently, the interfacial binding model including the appropriate binary SAM structure and the versatile streptavidin-biotin interaction was chosen as the basic supramolecular architecture for the fabrication of a SPFS-based immunoassay. Next, the affinity characteristics between different antibodies and hCG were measured via an equilibrium binding analysis, which is the first example for the titration of such a high affinity interaction by SPFS. The results agree very well with the constants derived from the literature. Finally, a sandwich assay and a competitive assay were selected as templates for SPFS-based hCG detection, and an excellent LOD of 0.15 mIU/ml was attained via the “one step” sandwich method. Such high sensitivity not only fulfills clinical requirements, but is also better than most other biosensors. Fully understanding how LHCII complexes transfer the sunlight energy directionally and efficiently to the reaction center is potentially useful for constructing biomimetic devices as solar cells. After the introduction of the structural and the spectroscopic features of LHCII, different surface immobilization strategies of LHCII were summarized next. Among them the strategy based on the His-tag and the immobilized metal (ion) affinity chromatography (IMAC) technique were of great interest and resulted in different kinds of home-fabricated His-tag chelating chips. Their substantial protein coupling capacity, maintenance of high biological activity and a remarkably repeatable binding ability on the same chip after regeneration was demonstrated. Moreover, different parameters related to the stability of surface coupled reconstituted complexes, including sucrose, detergent, lipid, oligomerization, temperature and circulation rate, were evaluated in order to standardize the most effective immobilization conditions. In addition, partial lipid bilayers obtained from LHCII contained proteo-liposomes fusion on the surface were observed by the QCM technique. Finally, the inter-complex energy transfer between neighboring LHCIIs on a gold protected silver surface by excitation with a blue laser (λ = 473nm) was recorded for the first time, and the factors influencing the energy transfer efficiency were evaluated.

Assembly and characterization of supramolecular architectures for biosensor applications

The research has included the efforts in designing, assembling and structurally and functionally characterizing supramolecular biofunctional architectures for optical biosensing applications. In the first part of the study, a class of interfaces based on the biotin-NeutrAvidin binding matrix for the quantitative control of enzyme surface coverage and activity was developed. Genetically modified ß-lactamase was chosen as a model enzyme and attached to five different types of NeutrAvidin-functionalized chip surfaces through a biotinylated spacer. All matrices are suitable for achieving a controlled enzyme surface density. Data obtained by SPR are in excellent agreement with those derived from optical waveguide measurements. Among the various protein-binding strategies investigated in this study, it was found that stiffness and order between alkanethiol-based SAMs and PEGylated surfaces are very important. Matrix D based on a Nb2O5 coating showed a satisfactory regeneration possibility. The surface-immobilized enzymes were found to be stable and sufficiently active enough for a catalytic activity assay. Many factors, such as the steric crowding effect of surface-attached enzymes, the electrostatic interaction between the negatively charged substrate (Nitrocefin) and the polycationic PLL-g-PEG/PEG-Biotin polymer, mass transport effect, and enzyme orientation, are shown to influence the kinetic parameters of catalytic analysis. Furthermore, a home-built Surface Plasmon Resonance Spectrometer of SPR and a commercial miniature Fiber Optic Absorbance Spectrometer (FOAS), served as a combination set-up for affinity and catalytic biosensor, respectively. The parallel measurements offer the opportunity of on-line activity detection of surface attached enzymes. The immobilized enzyme does not have to be in contact with the catalytic biosensor. The SPR chip can easily be cleaned and used for recycling. Additionally, with regard to the application of FOAS, the integrated SPR technique allows for the quantitative control of the surface density of the enzyme, which is highly relevant for the enzymatic activity. Finally, the miniaturized portable FOAS devices can easily be combined as an add-on device with many other in situ interfacial detection techniques, such as optical waveguide lightmode spectroscopy (OWLS), the quartz crystal microbalance (QCM) measurements, or impedance spectroscopy (IS). Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) allows for an absolute determination of intrinsic rate constants describing the true parameters that control interfacial hybridization. Thus it also allows for a study of the difference of the surface coupling influences between OMCVD gold particles and planar metal films presented in the second part. The multilayer growth process was found to proceed similarly to the way it occurs on planar metal substrates. In contrast to planar bulk metal surfaces, metal colloids exhibit a narrow UV-vis absorption band. This absorption band is observed if the incident photon frequency is resonant with the collective oscillation of the conduction electrons and is known as the localized surface plasmon resonance (LSPR). LSPR excitation results in extremely large molar extinction coefficients, which are due to a combination of both absorption and scattering. When considering metal-enhanced fluorescence we expect the absorption to cause quenching and the scattering to cause enhancement. Our further study will focus on the developing of a detection platform with larger gold particles, which will display a dominant scattering component and enhance the fluorescence signal. Furthermore, the results of sequence-specific detection of DNA hybridization based on OMCVD gold particles provide an excellent application potential for this kind of cheap, simple, and mild preparation protocol applied in this gold fabrication method. In the final chapter, SPFS was used for the in-depth characterizations of the conformational changes of commercial carboxymethyl dextran (CMD) substrate induced by pH and ionic strength variations were studied using surface plasmon resonance spectroscopy. The pH response of CMD is due to the changes in the electrostatics of the system between its protonated and deprotonated forms, while the ionic strength response is attributed from the charge screening effect of the cations that shield the charge of the carboxyl groups and prevent an efficient electrostatic repulsion. Additional studies were performed using SPFS with the aim of fluorophore labeling the carboxymethyl groups. CMD matrices showed typical pH and ionic strength responses, such as high pH and low ionic strength swelling. Furthermore, the effects of the surface charge and the crosslink density of the CMD matrix on the extent of stimuli responses were investigated. The swelling/collapse ratio decreased with decreasing surface concentration of the carboxyl groups and increasing crosslink density. The study of the CMD responses to external and internal variables will provide valuable background information for practical applications.

Polymer properties measured by micromechanical cantilever technique

Surface stress changes induced by specific adsorption of molecules were investigated using a micromechanical cantilever sensor (MCS) device. 16 MCS are grouped within four separate wells. Each well can be addressed independently by different liquid enabling functionalization of MCS separately by flowing different solutions through each well and performing sensing and reference experiments simultaneously. In addition, each well contains a fixed reference mirror, which allows measuring the absolute bending of MCS. The effect of the flow rate on the MCS bending change was found to be dependent on the absolute bending value of MCS. In addition, the signal from the reference mirror can be used to follow refractive index changes upon mixing different solutions. Finite element simulation of solution exchange in wells was compared with experiment results. Both revealed that one solution can be exchanged by another one after a total volume of 200 µl has flown through. Using MCS, the adsorption of thiolated deoxyribonucleic acid (DNA) molecules and 6-mercapto-1-hexanol (MCH) on gold surfaces, and the DNA hybridization were performed. The nanomechanical response is in agreement with data reported by Fritz et al.1 Thus, the multiwell device is readily applicable for sensing of multiple chemical and biological recognition events in a single step. In this context controlled release and uptake of drugs are currently widely discussed. As a model system, we have used polystyrene (PS) spheres with diameters in the order of µm. The swelling behavior of individual PS spheres in toluene vapor was studied via mass loading by means of micromechanical cantilever sensors. For 4–8% cross-linked PS a mass increase of 180% in saturated toluene vapor was measured. In addition, the diameter change in saturated toluene vapor was measured and the corresponding volume increase of 200% was calculated. The mass of the swollen PS sphere decreases with increasing exposure time to ultraviolet (UV) light. The swelling response is significantly different between the first and the second exposure to toluene vapor. This is attributed to the formation of a cross-linked shell at the surface of the PS spheres. Shape persistent parts were observed for locally UV irradiated PS spheres. These PS spheres were found to be fluorescent and cracks occur after exposure in toluene liquid. The diffusion time of dye molecules in PS spheres increases with increasing chemical cross-linking density. This concept of locally dissolving non cross-linked PS from the sphere was applied to fabricate donut structures on surfaces. Arrays of PS spheres were fabricated using spin coating. The donut structure was produced simply after liquid solvent rinsing. The complete cross-linking of PS spheres was found after long exposure time to UV. We found that stabilizers play a major role in the formation of the donut nanostructures.

Electrostatic supramolecular assembly of charged dendritic polymers and their biological application

The aim of this study was the development of functional multilayer films through electrostatic layer by layer (LbL) assembly of dendritic macromolecules, the investigation of the fundamental properties of these multilalyered films and the study of their biological applications. rnThe synthesis of the anionic hyperbranched polyglycerols (hbPG) and the preparation of multilayers made of hbPG/phosphorus dendrimer as well as the influences of deposition conditions on multilayers were reported. The thicknesses of multilayer films increase with a decrease of molecular weight of anionic hbPGs. The multilayer films fabricated by low molecular weight hbPGs grow less regularly due to the less charged carboxylic acid groups providing the relative weaker electrostatic forces for the deposition. The thicknesses of multilayer films are reduced with increasing pH values and decreasing the concentration of NaCl. The observed changes of multilayer thickness and surface morphology could be interpreted with the aid of theories regarding the charge density and conformation of the anionic hbPG chains in solution. rnBesides the study of fundamental properties of hbPG/phosphorus multilayer films, antifouling thin films derived from hbPG layers were developed. The antifouling properties of hbPG layers were found to correlate with factors of the molecular weight of anionic hbPG and the film thickness. It was demonstrated that anionic hbPG single layer with highest molecular weight can reduce non specific protein adsorption more efficiently than single layer with lower molecular weight and all the hbPG bilayers possessed excellent property of antifouling. rnPhosphorus dendrimer multilayers were successfully prepared as the platforms to detect DNA immobilization and hybridization. The effect of NaCl concentration on the multilayer film thickness was evaluated to obtain the optimized film thickness. Making use of the multilayer deposited at the optimized condition as a substrate, a high loading of DNA probes was achieved through covalent coupling of probe DNA with the as-formed multilayer films. The hybridization of target DNA with immobilized probe DNA was then carried out and studied by SPFS. The limit of detection upon hybridization was estimated on various dendrimer multilayer platforms. The minimum detection concentration for DNA hybridization is in the same order of magnitude compared with other neutral phosphorus dendrimer systems. Furthermore, the LbL deposition of phosphorus dendrimer multilayers provided a mild and simple way to prepare platforms as DNA microarrays. rnBased on the phosphorus dendrimer multilayer systems, dendritic star polymers were employed which have more reactive groups than that phosphorus dendrimers. The as-assembled dendritic star polymer multilayer films exhibited such distinct morphology characteristics that they underwent extensive structural reorganization upon post-treatment under different pH conditions. Kinetic binding of probe DNA molecules on the outermost negatively charged dendritic surface was studied by SPR as well. The binding capacities of probe DNA on the multilayer surfaces fabricated from the first-generation and the second-generation of dendritic star polymers were compared. The improved binding capacity was achieved from the second-generation of dendritic star polymer multilayer films due to their more reactive groups. DNA hybridization reaction on dendritic multilayer films was investigated by SPFS. The similar hybridization behaviors were found on both multilayer surfaces. Meanwhile, the hybridization kinetic affinities were compared with that of phosphorus dendrimer multilayer surfaces and showed improved detection sensitivity than phosphorus dendrimer multilayer films.rn

DNA block copolymers - synthesis, morphologies and applications

The last decades have witnessed significant and rapid progress in polymer chemistry and molecular biology. The invention of PCR and advances in automated solid phase synthesis of DNA have made this biological entity broadly available to all researchers across biological and chemical sciences. Thanks to the development of a variety of polymerization techniques, macromolecules can be synthesized with predetermined molecular weights and excellent structural control. In recent years these two exciting areas of research converged to generate a new type of nucleic acid hybrid material, consisting of oligodeoxynucleotides and organic polymers. By conjugating these two classes of materials, DNA block copolymers are generated exhibiting engineered material properties that cannot be realized with polymers or nucleic acids alone. Different synthetic strategies based on grafting onto routes in solution or on solid support were developed which afforded DNA block copolymers with hydrophilic, hydrophobic and thermoresponsive organic polymers in good yields. Beside the preparation of DNA block copolymers with a relative short DNA-segment, it was also demonstrated how these bioorganic polymers can be synthesized exhibiting large DNA blocks (>1000 bases) applying the polymerase chain reaction. Amphiphilic DNA block copolymers, which were synthesized fully automated in a DNA synthesizer, self-assemble into well-defined nanoparticles. Hybridization of spherical micelles with long DNA templates that encode several times the sequence of the micelle corona induced a transformation into rod-like micelles. The Watson-Crick motif aligned the hydrophobic polymer segments along the DNA double helix, which resulted in selective dimer formation. Even the length of the resulting nanostructures could be precisely adjusted by the number of nucleotides of the templates. In addition to changing the structural properties of DNA-b-PPO micelles, these materials were applied as 3D nanoscopic scaffolds for organic reactions. The DNA strands of the corona were organized by hydrophobic interactions of the organic polymer segments in such a fashion that several DNA-templated organic reactions proceeded in a sequence specific manner; either at the surface of the micelles or at the interface between the biological and the organic polymer blocks. The yields of reactions employing the micellar template were equivalent or better than existing template architectures. Aside from its physical properties and the morphologies achieved, an important requirement for a new biomaterial is its biocompatibility and interaction with living systems, i.e. human cells. The toxicity of the nanoparticles was analyzed by a cell proliferation assay. Motivated by the non-toxic nature of the amphiphilic DNA block copolymers, these nanoobjects were employed as drug delivery vehicles to target the anticancer drug to a tumor tissue. The micelles obtained from DNA block copolymers were easily functionalized with targeting units by hybridization. This facile route allowed studying the effect of the amount of targeting units on the targeting efficacy. By varying the site of functionalization, i.e. 5’ or 3’, the outcome of having the targeting unit at the periphery of the micelle or in the core of the micelle was studied. Additionally, these micelles were loaded with an anticancer drug, doxorubicin, and then applied to tumor cells. The viability of the cells was calculated in the presence and absence of targeting unit. It was demonstrated that the tumor cells bearing folate receptors showed a high mortality when the targeting unit was attached to the nanocarrier.

DNA-Polymer-Hybride als molekulare Bausteine

Die DNA hat sich durch die herausstechende Eigenschaft zur Selbstorganisation in den Naturwissenschaften zu einem beliebten Werkzeug entwickelt. In dieser Arbeit wurde die Oligonukleotidselbsterkennung zum Aufbau komplexer Multiblockcopolymere genutzt. Dabei dienten komplementäre einzelsträngige Oligonukleotidsequenzen (ssDNA) als adressierbare Verbindungsstücke zwischen synthetischen Blöcken. Als Bausteine wurden asymmetrische Dreiblockcopolymere der Form DNA1-Polymer-DNA2 aus einer flexiblen Polymereinheit (PEO bzw. PPO) die an beiden Enden mit unterschiedlichen Oligonukleotidsequenzen „funktionalisiert“ ist, verwendet. Diese Bausteine konnten durch die Kombination von Festphasensynthese der Oligonukleotide und Blockkopplung dargestellt werden. Die Oligonukleotidsequenzen wurden so gewählt, dass deren Hybridisierung zu einer bei Raumtemperatur stabilen Verbindung führt. Durch die Verwendung dieser Bausteine erhält man ein modulares System, dass sich durch seine hohe Flexibilität auszeichnet. Aus den dargestellten Dreiblockcopolymeren konnten verschiedene alternierende Multiblockcopolymere aufgebaut werden, wobei die Anzahl der Blöcke (von 11 bis 15) und das PEO / PPO- Verhältnis variiert wurden. Derartige Strukturen sind auf der Grundlage chemischer Synthesen unerreichbar. Die Flexibilität dieses modularen Systems konnte gezeigt werden, indem einzelne Blockbausteine zur Strukturaufklärung einfach ausgetauscht oder weggelassen werden konnten. Durch geeignete Wahl der DNA-Sequenzen konnte zusätzlich das Polymerisationsverhalten dieser Bauelemente untersucht werden. Die Integration längerer kettensteifer DNA-Abschnitte in die Multiblockstrukturen erfolgte durch die Verwendung teilkomplementärer Oligonukleotide. Diese bieten den Vorteil, dass bis zu einer Größe von etwa 150 bp sowohl die Länge als auch die Sequenz der Doppelstrangabschnitte und sticky-ends frei variiert werden können. Die biosynthetischen Dreiblockcopolymere dienten hier als Linkermoleküle zwischen den einzelnen dsDNA-Blöcken. Nach diesem Konzept wurde ein Nonamer als Modellsystem eines mehrfach gebrochenen Stäbchens synthetisiert. Außerdem wurden mit Hilfe der Polymerase Kettenreaktion (PCR) semiflexible DNA Abschnitte erzeugt. Durch die Wahl des Synthesewegs konnte sowohl die Länge der semiflexiblen Einheit als auch die Länge und die Sequenz des sticky-ends variiert werden. Anhand dieser Modellverbindungen wurde dann das Hybridisierungsverhalten in Abhängigkeit der Linker- und Segmentlängen untersucht.