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Schematic diagram of the 1695 bp long DNA fragment containing the target sequence for TFO-GAPDH and the two expected breakage fragments of 681 bp and 1024 bp length. The TFO is I-125-labeled at the 3′-terminal cytosine and binds to the polypurine target sequence in reverse orientation.
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Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA doubl...
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... e target binding site and induced breakage fragments of I-125-TFO-GAPDH are shown in Figure 3. Th e results of the DSB analysis revealed that the I-125-TFO-GAPDH induced a single DSB in the 1695 bp target fragment (Figure 4a, ) resulting in two expected breakage fragments of 1024 bp and 681 bp (Figures 4a, ). ...Similar publications
Arrays of oligonucleotides synthesized in the 5 0 ! 3 0 direction have potential benefit in several areas of life sciences research because the free 3 0 -end can be modified by enzymatic reactions. A Geniom One instrument (febit biomed GmbH, Germany), with integrated chip fabrication, multiplex primer extension, fluorescence imaging, and data analy...
Citations
... TFO are major groove-binding ligands, which are associated with the DNA duplex via hydrogen bonds known as Hoogsteen or reverse Hoogsteen hydrogen bonds [1]. In various studies [2][3][4] it was demonstrated that I-125 labeled TFO are able to induce DNA damage in a site directed manner. Furthermore, when the site specific DNA damage is located within a gene locus the respective gene expression was shown to be altered. ...
In this study we investigated the effects of I-125-TFO-BCL2 targeting a sequence located within intron 2 at 5422 bp downstream of the BCL2 promotor on gene expression, translocation frequency and protein expression of the human BCL2 gene.
... On the other hand, the radiosensitizing mechanism operating through DNA damage could be more efficient. A solution of this dilemma could be based on selective targeting of nanoparticles to specific genome sequences, like oncogenes, using appropriately designed oligo-nucleotides as being available for radio-emitters [70]. With techniques of COMBO-FISH [71,72] and PNA probe combinations [73], NPs may be transferred to cell nuclei and specifically addressed to given chromatin targets. ...
From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the “physics” behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2–3 nm). Next, we introduced a novel super-resolution approach—single molecule localization microscopy (SMLM)—to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.
... Similarly, Kotzerke et al. (2014) radiolabeled DAPI, a dual groove-binder, intercalator and commonly employed DNA dye, with 99m Tc. In related work, the anthracycline doxorubicin, a widely used topoisomerase II inhibitor and DNA intercalating chemotherapeutic, was conjugated to 99m Tc to enhance its Kotzerke et al., 2014 125 I-and 111 In-labeled TFOs Dahmen and Kriehuber, 2012;Dahmen et al., 2016 In-labeled anti-γH2AX antibody Cornelissen et al., 2012Cornelissen et al., , 2013 5-125 I-3'-O-(17β-succinyl-5α-androstan-3-one)-2'deoxyuridine monophosphate Kortylewicz et al., 2012;Han et al., 2014 Nucleus -trafficking cell surface receptors 111 In-labeled nimotuzumab Fasih et al., 2012 111 In-labeled hEGF Vallis et al., 2014 67 Ga-, 111 In-and 125 I-labeled MNT targeting EGFR, folate or melanocortin receptor Slastnikova et al., 2012Slastnikova et al., , 2017aKoumarianou et al., 2014 125 I-labeled monoclonal antibody 425 Quang and Brady, 2004 111 In-trastuzumab Costantini et al., 2007Costantini et al., , 2008 Methotrexate-loaded BCM conjugated to 111 In, an NLS, and trastuzumab Fab fragments potency and diagnostic potential (Imstepf et al., 2015). It was shown that the conjugate was readily taken up by the nucleus, caused extensive DNA damage, and exhibited a dose-dependent reduction in cell survival in several cancer cell lines. ...
... An additional option that has been explored for targeting of cancer cell DNA is the use of triplex forming oligonucleotides (TFO), site-specific molecules that bind to the major groove of duplex DNA to form a triplex helix. Several studies by Dahmen and Kriehuber (2012) and Dahmen et al. (2016Dahmen et al. ( , 2017 have demonstrated that TFOs can be readily labeled with radionuclides, such as 125 I and 111 In, and that these conjugates can exert site-and sequence-specific DNA damage in cancer cells. ...
The last decade has seen rapid growth in the use of theranostic radionuclides for the treatment and imaging of a wide range of cancers. Radionuclide therapy and imaging rely on a radiolabeled vector to specifically target cancer cells. Radionuclides that emit β particles have thus far dominated the field of targeted radionuclide therapy (TRT), mainly because the longer range (µm-mm track length) of these particles offsets the heterogeneous expression of the molecular target. Shorter range (nm-µm track length) α-and Auger electron (AE)-emitting radionuclides on the other hand provide high ionization densities at the site of decay which could overcome much of the toxicity associated with β-emitters. Given that there is a growing body of evidence that other sensitive sites besides the DNA, such as the cell membrane and mitochondria, could be critical targets in TRT, improved techniques in detecting the subcellular distribution of these radionuclides are necessary, especially since many β-emitting radionuclides also emit AE. The successful development of TRT agents capable of homing to targets with subcellular precision demands the parallel development of quantitative assays for evaluation of spatial distribution of radionuclides in the nm-µm range. In this review, the status of research directed at subcellular targeting of radionuclide theranostics and the methods for imaging and quantification of radionuclide localization at the nanoscale are described.
... The antigene strategy is based on the sequence-specific recognition of duplex DNA by triplexforming oligonucleotides (TFOs) at the major groove side, which can modulate gene expression at the transcriptional level. TFOs have been exploited in a wide range of biological activities (1)(2)(3)(4), including gene expression regulation (5)(6)(7), site-specific DNA damage (8)(9)(10)(11), DNA repair, recombination (12,13) and mutagenesis (14,15). ...
The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.
... Since the field of radiotherapy is still struggling to spare normal tissue while enhancing the dose deposition in malignant cells [5], especially the directed transport of damage sources into cancer cells is becoming more and more the focus of attention. Since the absorption of a photoelectron by particles with high atomic number Z can lead to the release of several Auger electrons [1,[6][7][8], the position as well as the distribution of those particles is a crucial parameter when it comes to increasing the therapeutic window. High energetic photoelectrons are long-ranged (several cm in water), and therefore do not allow a precise and well calibrated dose deposition within a tumor without harming healthy tissue substantially. ...
Localization microscopy has shown to be capable of systematic investigations on the arrangement and counting of cellular uptake of gold nanoparticles (GNP) with nanometer resolution. In this article, we show that the application of specially modified RNA targeting gold nanoparticles (“SmartFlares”) can result in ring like shaped GNP arrangements around the cell nucleus. Transmission electron microscopy revealed GNP accumulation in vicinity to the intracellular membrane structures including them of the endoplasmatic reticulum. A quantification of the radio therapeutic dose enhancement as a proof of principle was conducted with γH2AX foci analysis: The application of both—SmartFlares and unmodified GNPs—lead to a significant dose enhancement with a factor of up to 1.2 times the dose deposition compared to non-treated breast cancer cells. This enhancement effect was even more pronounced for SmartFlares. Furthermore, it was shown that a magnetic field of 1 Tesla simultaneously applied during irradiation has no detectable influence on neither the structure nor the dose enhancement dealt by gold nanoparticles.
... On the other hand, the radiosensitizing mechanism operating through DNA damage could be more efficient. A solution of this dilemma could be based on selective targeting of nanoparticles to specific genome sequences, like oncogenes, using appropriately designed oligo-nucleotides as being available for radio-emitters [70]. With techniques of COMBO-FISH [71,72] and PNA probe combinations [73], NPs may be transferred to cell nuclei and specifically addressed to given chromatin targets. ...
... These authors conclude that DSBs produced by 125 I-TFO exhibit a high degree of clustered base damages in the proximity of the DSB end and they assume that those damages may be a major factor leading to the less successful in vitro repair by the non-homologous end joining pathway (NHEJ). Additionally, 125 I-labelled TFO were shown to induce sequence specific complex DNA lesions and specific alterations of gene expression, underlining its capacity to significantly increase the number of DSBs in targeted genes [24]. ...
DNA-associated Auger electron emitters (AEE) cause cellular damage leading to high-LET type cell survival curves indicating an enhanced relative biological effectiveness. Double strand breaks (DSBs) induced by Iodine-125-deoxyuridine (125I-UdR) decays are claimed to be very complex. To elucidate the assumed genotoxic potential of 125I-UdR, chromatid aberrations were analysed in exposed human peripheral blood lymphocytes (PBL).
... However, the prerequisites are prepared and the tools for these developments are clear so that the dream is coming close to reality. DSBs could then be artificially introduced, for instance, by an oligonucleotide carrying an Auger electron emitter, 166 into specific structurally and functionally distinct chromatin domains. These could be labeled by a fluorescence point pattern of specific oligonucleotides and their behavior during repair could be followed in real time with living cells with the help of high-tech, live-cell, 3D multicolor nanoscopy. ...
Recent ground-breaking developments in Omics have generated new hope for overcoming the complexity and variability of biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and proteins interact in the frame of complex networks to preserve genome integrity has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Nuclear architecture and nuclear processes, including DNA damage responses, are precisely organized in space and time. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone, but requires sophisticated structural probing and imaging. Based on the results obtained from studying the relationship between higher-order chromatin structure, DNA double-strand break induction and repair, and the formation of chromosomal translocations, we show the development of Omics solutions especially for radiation research (radiomics) (discussed in this article) and how confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place the Omics data in the context of space and time (discussed in our other article in this issue, "Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part B-Structuromics"). Finally, we introduce a novel method of specific chromatin nanotargeting and speculate future perspectives, which may combine nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.
... However, the prerequisites are prepared and the tools for these developments are clear so that the dream is coming close to reality. DSBs could then be artificially introduced, for instance, by an oligonucleotide carrying an Auger electron emitter, 166 into specific structurally and functionally distinct chromatin domains. These could be labeled by a fluorescence point pattern of specific oligonucleotides and their behavior during repair could be followed in real time with living cells with the help of high-tech, live-cell, 3D multicolor nanoscopy. ...
Recent groundbreaking developments in Omics and bioinformatics have generated new hope for overcoming the complexity and variability of (radio)biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and dozens of proteins interact in the frame of complex signaling and repair pathways (or, rather, networks) to preserve the integrity of the genome has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone; it requires sophisticated structural probing and imaging. In the first part of this review, the article "Giving Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part A--Radiomics," we showed the development of different Omics solutions and how they are contributing to a better understanding of cellular radiation response. In this Part B we show how high-resolution confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place Omics data in the context of space and time. The dynamics of double-strand breaks during repair processes and chromosomal rearrangements at the microscale correlated to aberration induction are explained. For the first time we visualize pan-nuclear nucleosomal rearrangements and clustering at the nanoscale during repair processes. Finally, we introduce a novel method of specific chromatin nanotargeting based on a computer database search of uniquely binding oligonucleotide combinations (COMBO-FISH). With these challenging techniques on hand, we speculate future perspectives that may combine specific COMBO-FISH nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real-time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.
... However, the prerequisites are prepared and the tools for these developments are clear so that the dream is coming close to reality. DSBs could then be artificially introduced, for instance, by an oligonucleotide carrying an Auger electron emitter, 166 into specific structurally and functionally distinct chromatin domains. These could be labeled by a fluorescence point pattern of specific oligonucleotides and their behavior during repair could be followed in real time with living cells with the help of high-tech, live-cell, 3D multicolor nanoscopy. ...
