Local alternative DNA structures, such as cruciforms (CF), left-handed DNA, triplex and quadruplex (G4) structures, are known to exist in all organisms. Many proteins have been shown to exhibit CF-specific binding properties or G4-binding properties. We have developed easy accessible web tools for analyses of CF and G4 and analyzed their presence in various genomic datasets including human genome. Using various biophysical and molecular biological methods including AFM, EMSA, fluorescent anisotropy, CD, yeast isogenic transactivation assay we have evaluated DNA-protein binding properties. We have also analyzed interaction of p53 tumor suppressor with various DNA targets. A deeper understanding of DNA-protein interactions is an important component to consider in the post-genomic era.