Characterization of monomeric protein domains that bind specifically to a highly-conserved 100-Bp DNA target within rRNA genes

摘要

Proofs of concept have shown that chromosomal gene clusters encoding ribosomal RNA (rRNA) constitute gene delivery integration loci that are optimal for transgene expression. However, because homologous recombination is efficient to integrate DNA segments into these genes in animals, new molecular tools are required to construct systems able to target molecules in the immediate vicinity of the rRNA genes.We investigated the properties of several DNA binding domains (DBDs) able to recognize specifically a motif within a 100-bp region of the rRNA genes that is 99-100% conserved among eukaryotes. Our findings demonstrate that two Myb-like DBDs originating from the endonucleases encoded by R2 non-LTR retrotransposons are promising candidates since they i) specifically recognize, with high affinity, a 20-bp binding site located within the expected genomic rDNA target, ii) act as monomers, iii) contain a nuclearlocalization signal, iv) remain functional when fused to another domain and, v) do not alter the functionality of the protein to which they are fused. However, results obtained in vivo with several R2DBD fusions reveal that two properties remain to be engineered before these DBDs can be integrated into a molecular targeting system directed into rRNA genes. The first concerns the ability of R2DBD to locate within the nucleolus, the organelle in which the rRNA genes reside. The second is the tendency of R2DBD to accumulate in certain parts of the nuclei, which limits its diffusion within nuclei. Solutions are discussed to circumvent these current limitations. Our results supply important information concerning the R2DBD properties and the targeting of plasmid DNA within nuclei. They will need to be further analyzed from three aspects; the unexploited advantages of the R2DBDs, the possibilities and limitations of fusion peptides for targeting integrations of non-viral vector, and the alternatives to fusion peptides for targeting vectors.