Intracellular Distribution of TiO₂−DNA Oligonucleotide Nanoconjugates Directed to Nucleolus and Mitochondria Indicates Sequence Specificity

Abstract

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...

Synchrotron Radiation Spectroscopic Techniques as Tools for the Medicinal Chemist: Microprobe X-Ray Fluorescence Imaging, X-Ray Absorption Spectroscopy, and Infrared Microspectroscopy

“…Microprobe SRXRF has proven itself as a technique for providing unequalled information regarding the distribution of metal and metalloid-containing drugs in cells and tumours at subtoxic and therapeutic doses. [8,26,[31][32]38,[40][41]56] Targets such as the nucleus, [26,38] including the euchromatin [32] and heterochromatin [8] regions, nucleoli, [32,48,52] membranes, and mitochondria [52] can be identified as sites of localisation following studies of existing and potential therapeutic and diagnostic agents. The technique has been invaluable for confirming theories of drug actions (namely, the coordinative action of cisplatin and its analogues) [38][39] and for confirming concepts associated with the modes of action of designed therapeutics (namely, confirmation of drug penetration to the site of dense DNA).…”

“…[35,[52][53] Consequently, it is not surprising that microprobe SRXRF is becoming recognised as a technique for imaging intracellular nanoparticles in efforts to develop and confirm the capabilities of these agents. [35,52] An exciting study has been reported by Paunesku et al [52] whereby they aimed to produce DNA-targeting TiO 2 nanoparticles that could be excited, causing the electropositive holes of the semiconductor to be injected into the DNA, ultimately leading to DNA scission. The important role of microprobe SRXRF was to establish whether these nanoparticles could be taken up by the cells and retained at their target DNA sequence.…”

“…The important role of microprobe SRXRF was to establish whether these nanoparticles could be taken up by the cells and retained at their target DNA sequence. [52] Two DNA targets were studied, those of ribosomal RNA located in the nucleoli or the mitochondria. It was shown that the different nanoconjugates were retained in detectable quantities exclusively in the subcellular compartments that they were designed to target.…”

“…It was shown that the different nanoconjugates were retained in detectable quantities exclusively in the subcellular compartments that they were designed to target. [52] The authors also used microprobe SRXRF to show that inclusion of the contrast agent, Gd(III), into the nanoparticle to produce Gd(III)-modified DNA-TiO 2 nanoconjugates, also resulted in a structure capable of specific organelle targeting. [54] The interest in this result was that the complex possessed the added ability of being detectable by magnetic resonance.…”

Selective binding of oligonucleotide on TiO 2 surfaces modified by swift heavy ion beam lithography

“…Besides the main interest of TiO 2 as a substitutive and repairing material, this material offers interesting surface characteristics [2,3] suitable to improve commonly used biomedical applications in e.g. immunology, oncology and molecular biology [4][5][6]. Previous studies have focused on different aspects of the sintering process, phase, composition, micro-and nanostructure of TiO 2 for specific application as a biomaterial.…”