Allergic asthma is a chronic inflammatory disease of the airways that is characterized by reversible bronchial obstruction, bronchial hyperresponsiveness, mucosal edema and increased mucus secretion. The transcription factor GATA-3 has been shown to play a central role in the immune pathogenesis of allergic inflammatory responses due to its function in differentiation and activation of Th2 cells and its expression in eosinophils, mast cells and natural killer T cells. Thus, GATA-3 is considered an interesting therapeutic target for the treatment of allergic disorders including allergic asthma. Antisense technologies represent a novel class of therapeutic strategies that offer the possibility of early therapeutic intervention since, they directly interact with the mRNA of target proteins and subsequently cause specific inhibition of gene expression. A major class of antisense molecules is so-called DNAzymes, which are single-stranded RNA-cleaving deoxyoligonucleotides. Such molecules may be chemically modified in different ways which opens interesting possibilities for the optimization of this kind of antisense technology. In the present work, the basic structure of the GATA-3-specific DNAzyme hgd40 was modified by insertion of an inverted thymidine at the 3'-end, phosphothioate and 2'-O-methyl-group base modifications as well as implementation of so-called locked nucleic acids. The original DNAzyme and the developed variants were functionally characterized. In particular, the influence of the modifications on the accessibility of the target sequence and the catalytic efficacy were examined. Furthermore, dependency of catalytic activities of the DNAzyme variants on magnesium concentrations was analysed and finally, the GATA-3-DNAzyme variants were evaluated with regard to potential off-target effects in an in vitro system. Analysis of cleavage activity revealed, that all developed variants could cleave the GATA-3-cRNA except the DNAzyme with a 2'-O-methyl-group modification hgd40-OMe3 and the LNAzyme hgd40-L2. The DNAzyme variant hgd40-3M modified with an inverse thymidine at the 3'-end and the version hgd40-L3 showed similarly high or even slightly enhanced cleavage efficacies compared to the unmodified DNAzyme hgd40. With regard to magnesium dependency, all DNAzymes demonstrated high activities also at physiological magnesium concentrations suggesting that all variants might be active also in vivo. In terms of possible off-target effects it could be demonstrated in cell culture experiments, that none of the DNAzyme variants did significantly activate innate immune mechanisms, particularly through interaction with the toll-like receptor 9 (TLR-9). In conclusion, future use of modified versions of the GATA-3-specific DNAzyme hgd40 as a novel therapeutic strategy for the treatment of allergic asthma seems to be auspicious. This concept should be verified in further in vivo studies.