Deoxy DNA oligonucleotides hybridize to matching DNA sequences in cells, as established in the literature, depending on active transcription of the target sequence and local molarity of the oligonucleotide. We investigated the intracellular distribution of nanoconjugates composed of deoxy DNA oligonucleotides attached to TiO 2 nanoparticles, thus creating a locally increased concentration of the oligonucleotide. Two types of nanoconjugates, with oligonucleotides matching mitochondrial or nucleolar DNA, were specifically retained in mitochondria or nucleoli.The use of free nucleic acids as therapeutic agents was conceptualized at the same time when the first attempts in genetic engineering were successfully concluded. Notwithstanding the difficulties regarding cellular uptake and intracellular stability of nucleic acids, oligonucleotides were found to be capable of "finding" the matching genomic sequence and leading to the repair of the target sequence [1][2][3][4] . The formation of matching hybrids inside cells is modulated by processes of transcription and replication.Our interest lies in using a similar approach in order to develop an agent that can be triggered to cause not genomic DNA repair but the opposite-DNA damage extensive enough to cause gene inactivation. For this purpose we are developing TiO 2 nanoparticles (3-5 nm) conjugated to 1 to 5 single stranded DNA oligonucleotides (6-8 nm) via dopamine used as a bidentate enediol ligand for TiO 2 5. The semiconductor TiO 2 nanoparticles can be excited causing the electropositive holes to be injected into the DNA ultimately leading to DNA scission as we have shown in vitro 5, 6 .* to whom reprint requests should be addressed: g-woloschak@northwestern.edu. The first step in developing nanoparticles for intracellular DNA targeting is to assure that they have the capacity to be taken up by cells and retained at their target DNA sequence. NIH Public AccessSince it is very difficult to identify the precise position of a single gene DNA target inside cells, we decided to monitor targeting of genes located in specific subcellular compartments. Two such DNA targets with clearly defined subcellular locations are genes for ribosomal RNA (rDNA) located in nucleolus inside cell nucleus; and mitochondrial genes, located in mitochondria in cellular cytoplasm. Therefore, we decided to compare 1) oligonucleotide targeting ribosomal 18S RNA gene (R18) located on the satellite arms of chromosomes and dispersed in the area of nucleolus in the interphase nucleus; 2) oligonucleotide targeting the mitochondrial genomic sequence-NADH dehydrogenase subunit 2 gene (ND2), and 3) nanoparticles without attached oligonucleotide DNA. While the DNA-free nanoparticles have no target inside cells, nucleolar and mitochondrial nanoconjugates have similar number of targets per cell, albeit distributed differently. Ribosomal 18S gene is present in the genome in about 300 hundred copies 7 co-localized in one location-in nucleoli. The mitochondrial target is present in a few copies in e...

Although copper has been reported to influence numerous proteins known to be important for angiogenesis, the enhanced sensitivity of this developmental process to copper bioavailability has remained an enigma, because copper metalloproteins are prevalent and essential throughout all cells. Recent developments in x-ray optics at third-generation synchrotron sources have provided a resource for highly sensitive visualization and quantitation of metalloproteins in biological samples. Here, we report the application of x-ray fluorescence microscopy (XFM) to in vitro models of angiogenesis and neurogenesis, revealing a surprisingly dramatic spatial relocalization specific to capillary formation of 80 -90% of endogenous cellular copper stores from intracellular compartments to the tips of nascent endothelial cell filopodia and across the cell membrane. Although copper chelation had no effect on process formation, an almost complete ablation of network formation was observed. XFM of highly vascularized ductal carcinomas showed copper clustering in putative neoangiogenic areas. This use of XFM for the study of a dynamic developmental process not only sheds light on the copper requirement for endothelial tube formation but highlights the value of synchrotron-based facilities in biological research.copper chelation ͉ human microvascular endothelial cells ͉ infiltrating ductal breast carcinoma E ndogenous metals, such as Cu, Fe, and Zn, are subject to complex regulation in cellular systems. They are required as cofactors or regulators of numerous proteins (1) and yet, if present in overabundance, are toxic and expose the cellular environment to adventitious redox activity (2). This delicate balance is thought to be achieved by sequestration of these metals within their target proteins, metallochaperone systems, or distinct subcellular compartments (3). Although many proteins that handle transition metals within cells have been identified (4), our knowledge as to how metal content is dynamically regulated in eukaryotic cells is still limited. To what extent does regulation of the metal ion content of individual metalloproteins, mediated by protein-protein interactions, serve as an additional level of regulation of cellular metalloprotein activity? Could such regulation result in polarization of transition metal distribution throughout a cell during a biological process? To begin to explore such questions, we examined a cellular system whose biology is acutely sensitive to modulation by metals.