The external interference of avidin/biotin (A/B) ligand-receptor binding and helix-to-coil denaturation (melting) transition in short DNA fragments will be investigated by single molecule force spectroscopy (SMFS). Binding forces will be investigated in hydrogen-bond forming solvents glycerol and ethylene glycol, either mixed with water or pure. Both A/B and DNA binding is based on establishment of numerous hydrogen bonds, as well as on solvophobic effects. In water, DNA hydrogen bonds are formed between complementary bases, and the bases themselves are hydrophobic in character. Biotin forms a complex with avidin by several hydrophobic, hydrogen bonding and polar interactions of biotin with the amino-acids of avidin. Solvophobic interaction in glycerol and even more in ethylene glycol is weaker than hydrophobic in water and thus A/B binding is expected to be weakened. This assumption will be proven by performing SMFS experiments in Linz to which students from Zagreb will join in order to train in SMFS. Linz group will extend this study towards avidin mutants made available to them by partners from Finland. For DNA it was observed previously that the melting transition temperature is reduced due to nonaqueous solvent, however this was not checked by SMFS and we also plan to correlate SMFS result on DNA binding (in Zagreb, upon training by Linz group) with transport measurements of fragment diffusion and conductivity (to be performed in Zagreb) in organic solvent environment. In this environment the Bjerrum length is enhanced as the latter are weaker dielectrics than water. This reduces the Debye-Hückel charge screening length and enhances Manning condensation. As the phosphate charges repulsion on opposing strands is the primary DNA melting force these electrostatic effects should influence DNA stability. Students from Linz will join these studies as a vehicle to get acquainted with fluorescence correlation spectroscopy and its applicability in research of live cells. Finally, as glycerol and ethylene glycol are highly important in industry and in laboratory as cryoprotectants, we hope that our results will contribute to the understanding of the molecular mechanism of their action.

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