Biomolecules have a central role in two relatively new, fast-developing scientific disciplines, nanobiotechnology and bionanotechnology. The properties and the behaviour of natural biomolecules need to be well studied and characterized in order to use them in modern technical applications. Quite often a genetic or chemical modification is needed to tailor the biomolecule suitable for a specific application.

The aim of this doctoral thesis was to characterize and modify the properties of biomolecules in order to develop molecular tools for applications in nanobiotechnology and bionanotechnology. Avidin proteins were selected for modification as they are already widely used in biotechnology due to their extraordinary tight affinity for a small vitamin, D-biotin (Kd ~10-15 M). In this study, covalent ligand binding was engineered and applied to one binding site of a dual chain avidin (dcAvd). The resulting dcAvd-Cys enabled the subsequent attachment of two different kinds of molecules, thiol-reactive compounds and biotinylated compounds, to the same protein pseudotetramer. This molecule could be a valuable tool for the immobilization of molecules in a controlled way.

The biochemical and biophysical characterization as well as the high-resolution crystallographic 3D-structures of two bradavidins revealed some behavioral traits atypical for avidins. In the tetrameric wild type bradavidin, the C-terminal amino acid residues interact with the ligand-binding site of the neighbouring monomer, thus acting as an intersubunit intrinsic ligand. This finding led to the development of a bradavidin specific Brad-tag, which was demonstrated to be applicable as an affinity tag (Kd ~2.5 × 10-5 M). Most of the characterized avidins are tetramers or dimers, whereas bradavidin II exists in a dynamic oligomeric state depending on the environment. The weak oligomerization tendency of bradavidin II may be of use in the development of fusion proteins.

In addition to avidins, two biomolecular complexes were studied. First, the self-assembling property of deoxyribonucleic acid (DNA) was used to develop a defined sized, self-assembling DNA structure (B–A–B-complex) based on triple crossover (TX) tiles. Dielectrophoresis was used to trap and immobilize the B–A–B-complex between two gold-nanoelectrodes via thiol-gold bonding. The feasibility to functionalize the complex was demonstrated using streptavidin. The measured direct conductivity of the B–A–B-complex was insignificant, and therefore, the complex may find use as a scaffold material for the precise assembly of other components in molecular electronics. Second, the electrostatic interaction between chitosan and DNA was used to create chitosan-DNA nanoparticles. These were studied as potential carriers for gene-delivery applications. The nanoparticles were functionalized by fluorescent labels and targeting peptides. A cell culture analysis demonstrated that the nanoparticles can be internalized by cells.

In conclusion, this thesis study produced versatile biomolecular tools based on modified avidins, a self-assembling DNA-complex and chitosan-DNA nanoparticles. Although the developed tools may find use in somewhat different end-point applications ranging from biosensor surfaces to protein detection, and from molecular electronics to gene delivery, the methods and materials used were similar in all of the studies. Altogether, the studied and engineered biomolecules provide valuable knowledge for the future development of biomolecular tools.