Effect of Ionizing Radiation from Computed Tomography on Differentiation of Human Embryonic Stem Cells into Neural Precursors.

We studied the effect of radiation from computed tomography (CT) scans on differentiation of human embryonic stem cells (hESCs) into neuronal lineage. hESCs were divided into three radiation exposure groups: 0-dose, low-dose, or high-dose exposure. Low dose was accomplished with a single 15 mGy CT dose index (CTDI) CT scan that approximated the dose for abdominal/pelvic CT examinations in adults while the high dose was achieved with several consecutive CT scans yielding a cumulative dose of 500 mGy CTDI. The neural induction was characterized by immunocytochemistry. Quantitative polymerase chain reaction (qPCR) and Western blots were used to measure expression of the neuronal markers PAX6 and NES and pluripotency marker OCT4. We did not find any visible morphological differences between neural precursors from irradiated and non-irradiated cells. However, quantitative analyses of neuronal markers showed that PAX6 expression was reduced following exposure to the high dose compared to 0-dose controls, while no such decrease in PAX6 expression was observed following exposure to the low dose. Similarly, a statistically significant reduction in expression of NES was observed following high-dose exposure, while after low-dose exposure, a modest but statistically significant reduction in NES expression was only observed on Day 8 of differentiation. Further studies are warranted to elucidate how lower or delayed expression of PAX6 and NES can impact human fetal brain development.

Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines.

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

PPG peptide nucleic acids that promote DNA guanine quadruplexes.

Recent studies have shown that guanine-rich (G-rich) sequences with the potential to form quadruplexes might play a role in normal transcription as well as overexpression of oncogenes. Chemical tools that allow examination of the specific roles of G-quadruplex formation in vivo, and their association with gene regulation will be essential to understanding the functions of these quadruplexes and might lead to beneficial therapies. Properly designed peptide nucleic acids (PNAs) can invade G-rich DNA duplexes and induce the formation of a G-quadruplex in the free DNA strand. Replacing guanines in the PNA sequence with pyrazolo[3,4-d]pyrimidine guanine (PPG) nucleobases eliminates G-quadruplex formation with PNA and promotes invasion of the target DNA.

Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region.

Guanine-rich sequences are highly abundant in the human genome, especially in regulatory regions. Because guanine-rich sequences have the unique ability to form G-quadruplexes, these structures may play a role in the regulation of gene transcription. In previous studies, we demonstrated that formation of G-quadruplexes could be induced with peptide nucleic acids (PNAs). PNAs designed to bind the C-rich strand upstream of the human BCL2 gene promoted quadruplex formation in the complementary G-rich strand. However, the question whether G-quadruplex formation was essential for PNA invasion remained unanswered. In this study, we compared PNA invasion in the native and mutant, i.e. not forming G-quadruplex, BCL2 sequences and showed that G-quadruplex is required for effective PNA invasion into duplex DNA. This finding provides strong evidence for not only sequence-specific, but also quadruplex specific, gene targeting with PNA probes. In addition, we examined DNA-duplex invasion potential of PNAs of various charges. Using the gel shift assay, chemical probing and dimethyl sulfate (DMS) protection studies, we determined that uncharged zwitterionic PNA has the highest binding specificity while preserving efficient duplex invasion.

Whole-genome gene expression profiling reveals the major role of nitric oxide in mediating the cellular transcriptional response to ionizing radiation in normal human fibroblasts.

The indirect biological effects of ionizing radiation (IR) are thought to be mediated largely by reactive oxygen and nitrogen species (ROS and RNS). However, no data are available on how nitric oxide (NO) modulates the response of normal human cells to IR exposures at the level of the whole transcriptome. Here, we examined the effects of NO and ROS scavengers, carboxy-PTIO and DMSO, on changes in global gene expression in cultured normal human fibroblasts after exposures to gamma-rays, aiming to elucidate the involvement of ROS and RNS in transcriptional response to IR. We found that NO depletion dramatically affects the gene expression in normal human cells following irradiation with gamma-rays. We observed striking (more than seven-fold) reduction of the number of upregulated genes upon NO scavenging compared to reference irradiated cell cultures. NO scavenging in irradiated IMR-90 cells results in induction of p53 signaling, DNA damage and DNA repair pathways.

Effect of exposure to ionizing radiation on competitive proliferation and differentiation of hESC.

PURPOSE: We studied the effects of computed tomography (CT) scan irradiation on proliferation and differentiation of human embryonic stem cells (hESCs). It was reported that hESC is extremely radiosensitive; exposure of hESC in cultures to 1 Gy of ionizing radiation (IR) results in massive apoptosis of the damaged cells and, thus, they are eliminated from the cultures. However, after recovery the surviving cells proliferate and differentiate normally. We hypothesized that IR-exposed hESC may still have growth rate disadvantage when they proliferate or differentiate in the presence of non-irradiated hESC, as has been shown for mouse hematopoietic stem cells in vivo. MATERIALS AND METHODS: To study such competitive proliferation and differentiation, we obtained cells of H9 hESC line that stably express green fluorescent protein (H9GFP). Irradiated with 50 mGy or 500 mGy H9GFP and non-irradiated H9 cells (or vice versa) were mixed and allowed to grow under pluripotency maintaining conditions or under conditions of directed differentiation into neuronal lineage for several passages. The ratio of H9GFP to H9 cells was measured after every passage or approximately every week. RESULTS: We observed competition of H9 and H9GFP cells; we found that the ratio of H9GFP to H9 cells increased with time in both proliferation and differentiation conditions regardless of irradiation, i.e. the H9GFP cells in general grew faster than H9 cells in the mixtures. However, we did not observe any consistent changes in the relative growth rate of irradiated versus non-irradiated hESC. CONCLUSIONS: We conclude that population of pluripotent hESC is very resilient; while damaged cells are eliminated from colonies, the surviving cells retain their pluripotency, ability to differentiate, and compete with non-irradiated isogenic cells. These findings are consistent with the results of our previous studies, and with the concept that early in pregnancy omnipotent cells injured by IR can be replaced by non-damaged cells with no impact on embryo development.

Dynamics of the transcriptome response of cultured human embryonic stem cells to ionizing radiation exposure.

One of the key consequences of exposure of human cells to genotoxic agents is the activation of DNA damage responses (DDR). While the mechanisms underpinning DDR in fully differentiated somatic human cells have been studied extensively, molecular signaling events and pathways involved in DDR in pluripotent human embryonic stem cells (hESC) remain largely unexplored. We studied changes in the human genome-wide transcriptome of H9 hESC line following exposures to 1Gy of gamma-radiation at 2h and 16h post-irradiation. Quantitative real-time PCR was performed to verify the expression data for a subset of genes. In parallel, the cell growth, DDR kinetics, and expression of pluripotency markers in irradiated hESC were monitored. The changes in gene expression in hESC after exposure to ionizing radiation (IR) are substantially different from those observed in somatic human cell lines. Gene expression patterns at 2h post-IR showed almost an exclusively p53-dependent, predominantly pro-apoptotic, signature with a total of only 30 up-regulated genes. In contrast, the gene expression patterns at 16h post-IR showed 354 differentially expressed genes, mostly involved in pro-survival pathways, such as increased expression of metallothioneins, ubiquitin cycle, and general metabolism signaling. Cell growth data paralleled trends in gene expression changes. DDR in hESC followed the kinetics reported for human somatic differentiated cells. The expression of pluripotency markers characteristic of undifferentiated hESC was not affected by exposure to IR during the time course of our analysis. Our data on dynamics of transcriptome response of irradiated hESCs may provide a valuable tool to screen for markers of IR exposure of human cells in their most naive state; thus unmasking the key elements of DDR; at the same time, avoiding the complexity of interpreting distinct cell type-dependent genotoxic stress responses of terminally differentiated cells.

Stabilization of G-quadruplex in the BCL2 promoter region in double-stranded DNA by invading short PNAs.

Numerous regulatory genes have G-rich regions that can potentially form quadruplex structures, possibly playing a role in transcription regulation. We studied a G-rich sequence in the BCL2 gene 176-bp upstream of the P1 promoter for G-quadruplex formation. Using circular dichroism (CD), thermal denaturation and dimethyl sulfate (DMS) footprinting, we found that a single-stranded oligonucleotide with the sequence of the BCL2 G-rich region forms a potassium-stabilized G-quadruplex. To study G-quadruplex formation in double-stranded DNA, the G-rich sequence of the BCL2 gene was inserted into plasmid DNA. We found that a G-quadruplex did not form in the insert at physiological conditions. To induce G-quadruplex formation, we used short peptide nucleic acids (PNAs) that bind to the complementary C-rich strand. We examined both short duplex-forming PNAs, complementary to the central part of the BCL2 gene, and triplex-forming bis-PNAs, complementary to sequences adjacent to the G-rich BCL2 region. Using a DMS protection assay, we demonstrated G-quadruplex formation within the G-rich sequence from the promoter region of the human BCL2 gene in plasmid DNA. Our results show that molecules binding the complementary C-strand facilitate G-quadruplex formation and introduce a new mode of PNA-mediated sequence-specific targeting.

Structural polymorphism of intramolecular quadruplex of human telomeric DNA: effect of cations, quadruplex-binding drugs and flanking sequences.

G-quadruplex structures formed in the telomeric DNA are thought to play a role in the telomere function. Drugs that stabilize the G-quadruplexes were shown to have anticancer effects. The structures formed by the basic telomeric quadruplex-forming unit G(3)(TTAG(3))(3) were the subject of multiple studies. Here, we employ (125)I-radioprobing, a method based on analysis of the distribution of DNA breaks after decay of (125)I incorporated into one of the nucleotides, to determine the fold of the telomeric DNA in the presence of TMPyP4 and telomestatin, G-quadruplex-binding ligands and putative anticancer drugs. We show that d[G(3)(TTAG(3))(3)(125)I-CT] adopts basket conformation in the presence of NaCl and that addition of either of the drugs does not change this conformation of the quadruplex. In KCl, the d[G(3)(TTAG(3))(3)(125)I-CT] is most likely present as a mixture of two or more conformations, but addition of the drugs stabilize the basket conformation. We also show that d[G(3)(TTAG(3))(3)(125)I-CT] with a 5'-flanking sequence folds into (3+1) type 2 conformation in KCl, while in NaCl it adopts a novel (3+1) basket conformation with a diagonal central loop. The results demonstrate the structural flexibility of the human telomeric DNA; and show how cations, quadruplex-binding drugs and flanking sequences can affect the conformation of the telomeric quadruplex.

Effect of Ionizing Radiation on Transcriptome during Neural Differentiation of Human Embryonic Stem Cells.

Human embryonic brain development is highly sensitive to ionizing radiation. However, detailed information on the mechanisms of this sensitivity is not available due to limited experimental data. In this study, differentiation of human embryonic stem cells (hESCs) to neural lineages was used as a model for early embryonic brain development to assess the effect of exposure to low (17 mGy) and high (572 mGy) doses of radiation on gene expression. Transcriptomes were assessed using RNA sequencing during neural differentiation at three time points in control and irradiated samples. The first time point was when the cells were still pluripotent (day 0), the second time point was during the stage of embryoid body formation (day 6), and the third and final time point was during the stage of neural rosette formation (day 10). Analysis of the transcriptomes revealed neurodifferentiation in both the control and irradiated cells. Low-dose irradiation did not result in changes in gene expression at any of the time points, whereas high-dose irradiation resulted in downregulation of some major neurodifferentiation markers on days 6 and 10. Gene ontology analysis showed that pathways related to nervous system development, neurogenesis and generation of neurons were among the most affected. Expression of such key regulators of neuronal development as NEUROG1, ARX, ASCL1, RFX4 and INSM1 was reduced more than twofold. In conclusion, exposure to a 17 mGy low dose of radiation was well tolerated by hESCs while exposure to 572 mGy significantly affected their genetic reprogramming into neuronal lineages.

Duplex formation at the 5' end affects the quadruplex conformation of the human telomeric repeat overhang in sodium but not in potassium.

Human telomeres contain numerous copies of the (TTAGGG)(n).(AATCCC)(n) repeated sequence with multiple TTAGGG repeats in 3' single-stranded overhangs. Single-stranded oligonucleotides consisting of four TTAGGG repeats can fold into various intramolecular quadruplex structures stabilized by quartets of guanines. The quadruplex structures are believed to play a role in telomere functions and considered as targets for anticancer drug design. In an effort to create a more realistic model of telomeric DNA, we designed oligonucleotides containing a duplex region at the 5' end and four telomeric repeats in the 3' overhang. We applied CD spectroscopy and (125)I radioprobing to determine the conformation of the quadruplexes formed in the 3' overhangs. We found that in the presence of NaCl the conformation of the quadruplex changes with formation of the 5' duplex and depends on the position of the interface between the duplex and the 3' telomeric sequence. When the duplex region extended to the first T of the first TTAGGG repeat, both CD and radioprobing data are consistent with the parallel propeller conformation of the overhang. In the presence of KCl, formation of the duplex at the 5' end of DNA molecules did not change the fold of the quadruplex in the overhang which was interpreted as a mixture of two isomers of 3+1 conformation regardless of the duplex-overhang interface position. Our results demonstrate that the interface between the duplex and single-stranded overhang can affect the conformation of the telomeric quadruplex.

Labeling Peptide Nucleic Acids with Indium-111.

Peptide nucleic acids (PNA) are widely used DNA mimics that bind sequence specifically to single- and double-stranded nucleic acids. Hence they are of interest in the design of gene-targeted radiotherapeutics that could deliver radiodamage to designated DNA and/or RNA sites. Here I describe a procedure for incorporation of gamma-emitting radionuclide 111In into PNA oligomers. Diethylenetriaminepentaacetic acid (DTPA) was conjugated to a lysine-containing mixed-base PNA. 111In-labeled PNAs were obtained by chelation of PNA-DTPA conjugates with 111In3+ in an acidic aqueous solution.

Iodine-125 radioprobing of intramolecular quadruplex conformation of human telomeric DNA in the presence of cationic porphyrin TMPyP4.

PURPOSE: A repeated, non-coding, DNA sequence d(TTAGGG)(n) is present in the telomeric ends of all human chromosomes. These repeats can adopt multiple inter- and intra-molecular non-B-DNA conformations that may play an important role in biological processes. We applied (125)I -radioprobing to assess the conformation of the human telomeric DNA fragment in a complex with the quadruplex-specific drug - cationic porphyrin TMPyP4. MATERIAL AND METHODS: Synthetic DNA oligonucleotides containing the telomeric sequence were labeled with (125)I. The probability of DNA breaks caused by decay of (125)I is inversely related to the distance between the radionuclide and the sugar unit of the DNA backbone; hence, the conformation of the DNA backbone can be deduced from the distribution of breaks. RESULTS: The obtained data indicate that the telomeric oligonucleotides predominantly fold into an intramolecular quadruplex conformation in the presence of TMPyP4. We propose a mixed-type (3 + 1) conformation of telomeric quadruplex in a complex with the cationic porphyrin TMPyP4 in solution. Binding of the porphyrin overrides the counterion effect on quadruplex conformation. CONCLUSIONS: We have demonstrated that (125)I radioprobing can be successfully applied not only to determine folding in G-quadruplexes, but also to reveal the mode of quadruplex interaction with small ligands.

Effects of DNA-targeted ionizing radiation produced by 5-[125I]iodo-2'-deoxyuridine on global gene expression in primary human cells.

BACKGROUND: This study assesses the whole-genome gene expression changes in a panel of primary human cell lines in response to DNA damage mediated by decay of DNA-incorporated radioiodinated thymidine analog 5-[125I]iodo-2'-deoxyuridine (125I-IUdR). Three normal human cell lines of different origin, namely, gingival fibroblasts AG09319, fetal skin fibroblasts GM05388 and neonatal foreskin epidermal keratinocytes (NHFK) were used in this study. DNA molecules were radiolabeled by incubation of cells in culture in a medium supplemented with either 3.7 kBq/ml or 18.5 kBq/ml of 125I-IUdR for 24 h followed by incubation in IUdR-free medium for additional 24 hours. Each experiment was carried out in quadruplicate. 125I-IUdR uptake was monitored by measuring DNA-associated radioactivity. The whole-genome gene expression changes were evaluated using Agilent Human Whole Genome oligo microarrays containing 44,290 elements representing all known and predicted human genes. DNA microarray dataset was independently partially validated with quantitative real-time PCR (RT-PCR). RESULTS: AG09319 gingival cells in culture responded to 125I-IUdR treatment by changing the expression level of 335 genes in total, whereas under the same conditions GM05388 and NHFK cells differentially expressed 49 genes and 27 genes, respectively. However, for GM05388 cells the number of differentially expressed genes increases with the rise of 125I-IUdR concentrations in cell culture media. The key up-regulated biological processes in a chosen panel of cell lines concern the regulation of protein kinase activities and/or cell death. Genes repressed in response to 125I-IUdR treatment are involved in cytokinesis, M phase of the cell cycle, chromosome architecture and organization, DNA metabolism, DNA packaging, DNA repair and response to DNA damage. Despite the disparate nature of the gene patterns elicited by 125I-induced DNA damage among the different cell lines, the differentially expressed transcripts reveal strikingly non-random chromosomal distribution in all the cell lines we used. CONCLUSION: Our data suggest that DNA-targeted ionizing radiation produced by 125I-IUdR results in changes in expression of only a limited subset of genes in primary human cells. The responsive genes are distributed non-randomly among the chromosomes; and a significant fraction of them is p53-dependent in the transcriptional regulation.

Phosphorylation of histone H2AX in radiation-induced micronuclei.

DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to gamma radiation, the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei increases. The majority of the gamma-H2AX-positive micronuclei are centromere-negative. The number of gamma-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most gamma-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are gamma-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, gamma-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with gamma-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with gamma-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with gamma-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.

Effect of ionizing radiation on the proliferation of human embryonic stem cells.

We studied the effect of ionizing radiation (IR) on continuous growth of seven hESC lines. Cells were exposed to 0, 0.2, or 1 Gy of X-rays, and the growth rates of cell populations were assessed by measuring areas of the same individual colonies versus time. The population doubling times (DT) of sham-irradiated cells varied from 18.9 to 28.7 hours for different cell lines. All cell lines showed similar reaction to IR, i.e. cell populations dropped within 24-48 hours post IR; after that they recovered and grew with the same rate as the sham-irradiated cells. The relative cell survival (RCS), i.e. the ratio of normalized cell population in the irradiated samples to that of the sham-irradiated ones varied from 0.6 to 0.8 after 0.2 Gy, and from 0.1 to 0.2 after 1 Gy IR for different cell lines. We found that the RCS values of hESC lines correlated directly with their DT, i.e. the faster cells grow the more radiosensitive they are. We also found that DT and RCS values of individual colonies varied significantly within all hESC lines. We believe that the method developed herein can be useful for assessing other cytotoxic insults on cultures of hESC.

Single nucleotide variations in cultured cancer cells: Effect of mismatch repair.

We assessed single nucleotide variations (SNVs) between individual cells in two cancer cell lines; DU145, from brain metastasis of prostate tumor with deficient mismatch repair; and HT1080, a fibrosarcoma cell line. Clones of individual cells were isolated, and sequenced using Ion Ampliseq comprehensive cancer panel that covered the exomes of 409 oncogenes and tumor suppressor genes. Five clones of DU145 and four clones of HT1080 cells were analyzed. We found from 7 to 12 unique SNVs between DU145 clones, while HT1080 clones showed no more than one unique SNV. We then sub-cloned individual cells from some of these isolated clones of DU145 and HT1080 cells. The sub-clones were expanded from a single cell to approximately one million cells after about 20 cell divisions. The sub-clones of DU145 cells had from one to four new unique SNVs within the sequenced regions. No unique SNVs were found between sub-clones of HT1080 cells. Our data demonstrate that the extent of genetic variation at the single nucleotide level in cultured cancer cells is significantly affected by the status of the DNA mismatch repair system.

Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts.

That irradiated cells affect their unirradiated 'bystander' neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named gamma-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces gamma-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple gamma-H2AX foci. After 18 h coculture with cells irradiated with 20 alpha-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with gamma-rays, and in cultures containing media conditioned on gamma-irradiated cells. DNA DSB repair proteins accumulated at gamma-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying gamma-H2AX focus formation may be responsible for its downstream manifestations.

Intramolecular quadruplex conformation of human telomeric DNA assessed with 125I-radioprobing.

A repeated non-coding DNA sequence d(TTAGGG)n is present in the telomeric ends of all human chromosomes. These repeats can adopt multiple inter and intramolecular non-B-DNA conformations that may play an important role in biological processes. Two intramolecular structures of the telomeric oligonucleotide dAGGG(TTAGGG)3, antiparallel and parallel, have been solved by NMR and X-ray crystallography. In both structures, the telomeric sequence adopts an intramolecular quadruplex structure that is stabilized by G-4 quartets, but the ways in which the sequence folds into the quadruplex are different. The folds of the human telomeric DNA were described as an anti-parallel basket-type and a parallel propeller-type. We applied 125I-radioprobing to determine the conformation of the telomeric quadruplex in solution, in the presence of either Na+ or K+ ions. The probability of DNA breaks caused by decay of 125I is inversely related to the distance between the radionuclide and the sugar unit of the DNA backbone; hence, the conformation of the DNA backbone can be deduced from the distribution of breaks. The probability of breaks measured in the presence of Na+ and K+ were compared with the distances in basket-type and propeller-type quadruplexes obtained from the NMR and crystal structures. Our radioprobing data demonstrate that the antiparallel conformation was present in solution in the presence of both K+ and Na+. The preferable conformation in the Na+-containing solution was the basket-type antiparallel quadruplex whereas the presence of K+ favored the chair-type antiparallel quadruplex. Thus, we believe that the two antiparallel and the parallel conformations may coexist in solution, and that their relative proportion is determined by the type and concentration of ions.

Site-specific strand breaks in RNA produced by (125)I radiodecay.

Decay of (125)I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of (125)I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3'-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when (125)I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that (125)I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.