TitleStructure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases active, two subunits of these zinc finger nucleases (ZFN) are typically assembled at the 5 cleavage site. Presumably due to cleavage at off-target sites, the use of ZFNs is often associated with significant cytotoxicity. Here, we describe a structure-based approach to reduce ZFN-induced toxicity. Rational redesign of the FokI dimer interface aiming at destabilizing dimerization in combination with preventing homodimerization of the ZFN subunits was based on protein modeling and energy calculations. Cell-based recombination 10 assays confirmed that the modified ZFNs elicit significantly reduced cytotoxicity without compromising on performance. Our results present a critical step towards the therapeutic application of the ZFN technology.

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The main transcriptional regulator of the human immunodeficiency virus, the Tat protein, recognizes and binds to a small structured RNA element at the 5' end of every viral mRNA, termed TAR. On the basis of published structural data of the molecular interactions between TAR and Tat-related peptides, we defined requirements for potential low-molecular weight inhibitors of TAR recognition by the Tat protein. In accordance with the resulting concept, a series of compounds was synthesized. In vitro evaluation of their potential to directly interfere with Tat-TAR interaction was used to define a new chemical class of potent Tat antagonistic substances. The most active compound competed with Tat-TAR complexation with a competition dose CD50 of 22 nM in vitro and blocked HIV expression in a cellular Tat transactivation system with an IC50 of 1.2 microM. The close relation between structural features of the interaction between TAR and a new type of inhibitory agent, "In-PRiNts" (for inhibitor of protein-ribonucleotide sequences), such as CGP 40336A and those of the Tat-TAR complex was confirmed by RNase A footprinting and by two-dimensional NMR. Structural implications for the complex between this class of compounds and TAR RNA will be presented.

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Background: Copper/zinc superoxide dismutase (SOD1) genetic mutants are associated with familial amyotrophic lateral sclerosis (ALS). Mutant proteins form abnormal aggregates. Results: We used imaging of live cells to observe SOD1 proteins harboring mutations associated with ALS. Conclusion: SOD1 mutations impair its dimerization, leading to subsequent aggregation. Significance: Analysis of the SOD1 quaternary structure in living human cells correlates with previous biochemical data.