Applications of Chromophores and Multiphoton Techniques to Study Structure and Interactions of Bio-macromolecules in Assembled State

Doctoral thesis, 2013

The research presented in this thesis is concerned with the linear and nonlinear optical properties of biopolymers and the chromophores that bind to them. This thesis combines analyses of the interactions of biomolecules with technological improvements of already existing systems for bionanotechnology-related research. The importance of precise control of biosystems is essential in elucidating the fundamental properties of biomolecules, such as DNA and amyloid fibrils, or biomolecule-dye adducts. A starting point for such studies is to examine the structures of DNA oligonucleotides loaded either in a polymeric carrier or water-based buffers. The DNA secondary structure as a function of relative humidity reveals a strong dependence on polyvinyl alcohol (PVA) hydration level, which is of relevance for nanotechnological studies of DNA-based supramolecular systems. PVA gel systems provide possibilities to test models of nucleic acids interactions and distributions in cellular contexts, including the structural stability of the genetic material in the cell and PVA-based packaging for gene delivery. A method by which duplex oligonucleotides, which contain sequences designed to provide specific binding sites, become amenable to polarised-light spectroscopy opens up new possibilities for studying the structures of DNA complexes that contain small adduct molecules, as well as proteins. However, the polymer environment strongly destabilises the DNA-dye complex. A study of DNA-dye and PVA-dye interactions was carried out using a homologous set of dyes from the cyanine family while gradually increasing the charge and DNA affinity. The successful orientation in PVA of the ruthenium dimer [μ-(11,11′-bidppz)(phen)4Ru2]4+, which was bound by threading intercalation to DNA oligonucleotide duplex hairpins, reveals that binding modes depend both on the oligonucleotide sequence and the chirality of the probe. The enantioselective binding properties of sterically rigid DNA probes, such as the studied ruthenium complex, can be used to increase the targeting specificities of short nucleic acids sequences, e.g., to inhibit transcription in a therapeutic context, such as the treatment of malaria or cancer. Moreover, ruthenium(II) complexes exhibit strong multiphoton absorption properties, discovered and quantified using a nonlinear spectroscopy Z-scan technique. In particular, the [(11,11′-bidppz)(phen)4Ru2]4+ complex was found to exhibit very strong two- and three-photon absorption properties, which were enhanced by substitution at the para position in the dimer structure; these properties are not commonly observed in flexible dimer chromophores, such as the ethidium homodimer. Metal-organic complexes may represent a new generation of DNA- and amyloid fibril-staining agents that have the advantage of exhibiting strong nonlinear optical properties. Labelling with organic dyes is also a strategy for visualising aggregated states of proteins and there is a growing need for more specific and photostable binding chromophores. The binding of dimeric ruthenium complexes and a stilbene derivative to amyloid fibrils was examined in the context of applying multiphoton-based technologies for diagnostic purposes. Interestingly, the aggregated states of misfolded proteins exhibit remarkable multiphoton absorption properties, most probably due to cooperative mechanisms that involve aromatic amino acids that are densely packed in the β-sheet, rod-shaped structures of fibrils. These types of self-assembling bio-derived nanomaterials that exhibit specific nonlinear properties may be valuable in various applications, ranging from bio-imaging technology to photonics.