DNA Secondary Structure Research Articles

Agnostic detection of genomic alterations by holistic DNA structural interrogation.

There is an established relationship between primary DNA sequence, secondary and tertiary chromatin structure, and transcriptional activity, suggesting that observed differences in one of these properties may reflect changes in the others. Here, we exploit these relationships to show that variations in DNA structure can be used to identify a wide range of genomic alterations in mammalian samples. In this proof-of-concept study we characterized and compared genome-wide histone occupancy by ChIP-Seq, DNA accessibility by ATAC-Seq, and chromosomal conformation by Hi-C for five CRISPR/Cas9-modified mammalian cell lines and their unmodified parent strains, as well as in one modified tissue sample and its parent strain. The results showed that the impact of genomic alterations on each of the levels of DNA organization varied depending on mutation type (insertion or deletion), size, and genomic location. The largest genomic alterations we identified included chromosomal rearrangements and deletions (greater than 200 Kb) in four of the modified cell lines, which can be difficult to identify by standard whole genome sequencing analysis. This multi-level DNA organizational analysis provides a sensitive approach for identifying a wide range of genomic and epigenomic perturbations that can be utilized for biomedical and biosecurity applications.

HERC2 Facilitates BLM and WRN Helicase Complex Interaction with RPA to Suppress G-Quadruplex DNA.

BLM and WRN are RecQ DNA helicasesessential for genomic stability. Here, we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacted with BLM, WRN, and replication protein A (RPA) complexes during the S-phase of the cell cycle. Depletion of HERC2 dissociated RPA from BLM and WRN complexes and significantly increased G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4-suppressing function. In vitro, HERC2 released RPA onto single-stranded DNA (ssDNA) rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 inhibited ubiquitination of RPA2, caused RPA accumulation in the helicase complexes, and increased G4, indicating an essential role for E3 activity in the suppression of G4. Both depletion of HERC2 and inactivation of E3 sensitized cells to the G4-interacting compounds telomestatin and pyridostatin. Overall, these results indicate that HERC2 is a master regulator of G4 suppression that affects the sensitivity of cells to G4 stabilizers. Given that HERC2 expression is frequently reduced in many types of cancers, G4 accumulation as a result of HERC2 deficiency may provide a therapeutic target for G4 stabilizers.Significance: HERC2 is revealed as a master regulator of G-quadruplex, a DNA secondary structure that triggers genomic instability and may serve as a potential molecular target in cancer therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6371/F1.large.jpg Cancer Res; 78(22); 6371-85. ©2018 AACR.

OA257] Influence of nonionizing millimeter electromagnetic radiation on tumor and healthy DNA

Purpose The present study was undertaken to investigate whether millimeter range electromagnetic nonionizing radiation (NIR) of selective frequencies can suppress tumor cells growth in vivowithout cytostatic agents. Methods The course of influence of NIR started 3 days before transplantation in order to raise activity of the animals immune system. On the fourth day animals were injected by sarcoma-37 and daily exposure was continued during 15 days. For study of the effect of irradiation on the secondary structure of DNA, in the experiments DNA isolated from the liver of healthy mice (hDNA) as well as from the tumor sarcoma 37 (tDNA) was used. Results After 15 sessions of exposure without cytostatics, at animals of the irradiated 0,5 h was observed an inhibition of tumor growth by 33.5% compared with a control group and a sharp suppression of the level of DNA methylation in 2.1 times. The tDNA has the high level of methylation (4.7 mol%), which after 0.5 h daily exposure becomes (2.2 mol%) close to the corresponding value for hDNA (1.9 mol%). Differential melting curves (DMC) of tDNA are shifted relatively DMC of the hDNA to lower temperatures, and in the DMC of tDNA the additional peaks are appeared. The obtained results are correlated with the spectrophotometric data. Under the influence of millimeter NIR the values of temperature and interval of melting of tDNA are changed and approach to the corresponding values of hDNA. Conclusions Hypermethylation of tumor DNA may cause a selective sensitivity of malignant cells toward the influence of the NIR which allows an expressed antitumor effect. It is possible, that 30 min daily exposure leads to the activation of specific molecular mechanisms of cells, resulting in a decrease of undesirable structural changes in the tumor DNA and inhibition of tumor growth. Presented preliminary results have demonstrated the promising application of millimeter NIR for clinical oncology in the treatment of malignancies without damaging other tissues and antitumoral drugs and without harmful ionizing radiotheraphy.

Alternative DNA secondary structure formation affects RNA polymerase II promoter-proximal pausing in human

BackgroundAlternative DNA secondary structures can arise from single-stranded DNA when duplex DNA is unwound during DNA processes such as transcription, resulting in the regulation or perturbation of these processes. We identify sites of high propensity to form stable DNA secondary structure across the human genome using Mfold and ViennaRNA programs with parameters for analyzing DNA.ResultsThe promoter-proximal regions of genes with paused transcription are significantly and energetically more favorable to form DNA secondary structure than non-paused genes or genes without RNA polymerase II (Pol II) binding. Using Pol II ChIP-seq, GRO-seq, NET-seq, and mNET-seq data, we arrive at a robust set of criteria for Pol II pausing, independent of annotation, and find that a highly stable secondary structure is likely to form about 10–50 nucleotides upstream of a Pol II pausing site. Structure probing data confirm the existence of DNA secondary structures enriched at the promoter-proximal regions of paused genes in human cells. Using an in vitro transcription assay, we demonstrate that Pol II pausing at HSPA1B, a human heat shock gene, is affected by manipulating DNA secondary structure upstream of the pausing site.ConclusionsOur results indicate alternative DNA secondary structure formation as a mechanism for how GC-rich sequences regulate RNA Pol II promoter-proximal pausing genome-wide.

In Silico Identification of Piperidinyl-amine Derivatives as Novel Dual Binders of Oncogene c-myc/c-Kit G-quadruplexes.

In the last years, it has been shown that the DNA secondary structure known as G-quadruplex is also involved in the regulation of oncogenes transcription, such as c-myc, c-Kit, KRAS, Bcl-2, VEGF, and PDGF. DNA G-quadruplexes, formed in the promoter region of these proto-oncogenes, are considered alternative anticancer targets since their stabilization causes a reduction of the related oncoprotein overexpression. In this study, a structure-based virtual screening toward the experimental DNA G-quadruplex structures of c-myc and c-Kit was performed by using Glide for the docking analysis of a commercial library of approximately 693 000 compounds. The best hits were submitted to thermodynamic and biophysical studies, highlighting the effective stabilization of both G-quadruplex oncogene promoter structures for three N-(4-piperidinylmethyl)amine derivatives, thus proposed as a new class of dual G-quadruplex binders.

RiboSketch: versatile visualization of multi-stranded RNA and DNA secondary structure.

Creating clear, visually pleasing 2D depictions of RNA and DNA strands and their interactions is important to facilitate and communicate insights related to nucleic acid structure. Here we present RiboSketch, a secondary structure image production application that enables the visualization of multistranded structures via layout algorithms, comprehensive editing capabilities, and a multitude of simulation modes. These interactive features allow RiboSketch to create publication quality diagrams for structures with a wide range of composition, size and complexity. The program may be run in any web browser without the need for installation, or as a standalone Java application. https://rnastructure.cancer.gov/ribosketch.

Study of DNA interaction with cobalt disulfophthalocyanine

DNA interaction with disulfophthalocyanine of cobalt (CodSPC) was studied by UVVIS spectrophotometry and low gradient viscometry. The thermal denaturation of DNA in complexes with CodSPC was compared with free DNA denaturation. The small increase in melting temperature indicates that the secondary structure of DNA in complexes is stable. As a results of experimental research the external binding of CodSPC to DNA was determined. The only one type of binding is accompanied by stacking of heterocycles at high concentrations of CodSPC. An increase in viscosity of DNA solutions with the growth of CodSPC concentration can be explained by intermolecular DNA contacts via CodSPC.

DNA Damage Induced by Gamma-Radiation Revealed from UV Absorption Spectroscopy

DNA damages are the main cause of radiation-induced cell death, mutations and carcinogenesis. The study of influence of environmental conditions on the radiation damage of DNA structure is important for the wide range of medical problems. The present work is devoted to the study of alterations in DNA structure caused by γ-irradiation at the variation of counterions concentration and dose rate.The DNA solutions of the ionic strengths 0.005M and 0.15M NaCl were exposed to γ-radiation with the doses of 0-100 Gy, dose rates2.3Gy/s and 0.3 Gy/s. The lesions in DNA structure (base damages, partial denaturation) were studied by the methods of UV absorption spectroscopy and DNA melting.Radiation-induced base damages decrease at the rise of NaCl concentration in the solution and at the lowering of the dose rate. The stability of DNA secondary structure increase at the rise of ionic strength of irradiated solution. The influence of radiation dose rate on the DNA helicity reveals definitely only at the dose of 100 Gy. The relative fall of the DNA melting temperatureafter the irradiation slightly depends on the dose rate and NaCl concentration.

Mapping three guanine oxidation products along DNA following exposure to three types of reactive oxygen species.

Reactive oxygen and nitrogen species generated during respiration, inflammation, and immune response can damage cellular DNA, contributing to aging, cancer, and neurodegeneration. The ability of oxidized DNA bases to interfere with DNA replication and transcription is strongly influenced by their chemical structures and locations within the genome. In the present work, we examined the influence of local DNA sequence context, DNA secondary structure, and oxidant identity on the efficiency and the chemistry of guanine oxidation in the context of the Kras protooncogene. A novel isotope labeling strategy developed in our laboratory was used to accurately map the formation of 2,2-diamino-4-[(2-deoxy-β-D-erythropentofuranosyl)amino]- 5(2 H)-oxazolone (Z), 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG), and 8-nitroguanine (8-NO2-G) lesions along DNA duplexes following photooxidation in the presence of riboflavin, treatment with nitrosoperoxycarbonate, and oxidation in the presence of hydroxyl radicals. Riboflavin-mediated photooxidation preferentially induced OG lesions at 5' guanines within GG repeats, while treatment with nitrosoperoxycarbonate targeted 3'-guanines within GG and AG dinucleotides. Little sequence selectivity was observed following hydroxyl radical-mediated oxidation. However, Z and 8-NO2-G adducts were overproduced at duplex ends, irrespective of oxidant identity. Overall, our results indicate that the patterns of Z, OG, and 8-NO2-G adduct formation in the genome are distinct and are influenced by oxidant identity and the secondary structure of DNA.

Micronomicin/tobramycin binding with DNA: fluorescence studies using of ethidium bromide as a probe and molecular docking analysis

Two aminoglycosides, micronomicin (MN), and tobramycin (TB), binding with DNA were studied using various spectroscopic techniques including fluorescence, UV–Vis, FT-IR, and CD spectroscopy coupled with relative viscosity and molecular docking. Studies of fluorescence quenching and time-resolved fluorescence spectroscopy all revealed that MN/TB quenching the fluorescence of DNA–EB belonged to static quenching. The binding constants and binding sites were obtained. The values of ΔH, ΔS, and ΔG suggested that van der Waals force or hydrogen bond might be the main binding force. FT-IR and CD spectroscopy revealed that the binding of MN/TB with DNA had an effect on the secondary structure of DNA. Binding mode of MN/TB with DNA was groove binding which was ascertained by viscosity measurements, CD spectroscopy, ionic strength, melting temperature (Tm), contrast experiments with single stranded (ssDNA), and double stranded DNA (dsDNA). Molecular docking analysis further confirmed that the groove binding was more acceptable result.

Characterization of the secondary structure and protein binding affinity of an intramolecular DNA four‐way junction.

Oligodeoxynucleotides (ODN) offer great promise as antigene and antisense reagents due to their high binding specificity, straight‐forward synthesis and low toxicity. Research shows that ODNs can also target proteins such as the DNA‐binding cytokine, High Mobility Group B1 (HMGB1). HMGB1 was initially classified as a highly abundant architectural nuclear protein. It is now clear that HMGB1 also functions as a redox sensitive damage associated molecular pattern (DAMP) molecule. And several studies link deleterious HMGB1 inflammatory signaling with an array of diseases and immune disorders such as: atherosclerosis, cystic fibrosis, lupus and sepsis. Hence, HMGB1 is gathering a great deal of attention as a biomarker and therapeutic target. We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. In order to enhance the stability of 4WJs for ex vivo studies, the helical termini of a DNA junction are modified to generate the intramolecular construct, i‐J1. The secondary structure, thermostability and protein binding affinity of i‐J1 are investigated vs. a control 4WJ (J1) and the intramolecular 4WJ, J4. Non‐denaturing gel electrophoresis assays show that i‐J1 possesses a global structural conformation that is analogous to the control 4WJ. Circular dichroism (CD) studies show that the secondary structure of i‐J1 is composed of B‐ and A‐form DNA helices. Moreover, the melting temperature (Tm) of i‐J1 is significantly higher than J1. The Tm of i‐J1 is ~ 30°C higher than J1. Finally, electrophoretic mobility shift assays (EMSAs) show that the HMG protein, HMGB1b, binds i‐J1 with nearly identical affinity to J1. The data clearly shows that the junction lattice of a DNA four‐way junction can be altered to enhance conformational stability and retain protein binding affinity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent.

Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

Pac1 Signals of Human Herpesviruses Contain a Highly Conserved G-Quadruplex Motif.

Packaging signals ( pac1 and pac2) of human herpesviruses (HHVs) that contain GC-rich elements are essential for cleavage and packaging of the virus. Here, we report the presence of putative G-quadruplex sequences (PQSs) in the packaging signal ( pac1) of all HHVs. Importantly, the residues critical for the formation of G-quadruplex structures were highly conserved as compared to those not critical for the formation of this DNA secondary structure, indicating that G-quadruplexes are positively selected within pac1 in the evolution of herpesviruses. CD spectroscopy, NMR spectroscopy, native/denaturing gel, and DMS footprinting confirmed the formation of G-quadruplex structures in all pac1 PQS oligonucleotides analyzed; the majority of the PQS had the propensity to form intermolecular structures. The presence of highly conserved G-quadruplex motifs at genomic locations critical for virus packaging has not been previously recognized. Our findings provide a new perspective on the putative functions of G-quadruplexes in virus genomes.

Electrochemical Monitoring of the Reversible Folding of Surface-Immobilized DNA i-Motifs.

Two cytosine (C) rich DNA sequences folding in i-motif upon protonation of C at low pH have been immobilized at gold electrodes to study the impact of the electrode|electrolyte interface on the stability of the noncanonical DNA secondary structure. The effects of the molecular composition and environment on the melting and folding of the structures immobilized at the gold surface have been compared to the properties of the DNA strands in solution. The DNA folding into i-motif upon protonation, both at the surface and in solution, results in a significant variation of the charge density which is monitored electrochemically through the electrostatic interactions between the DNA strand and the electroactive hexaammineruthenium(III). This method is shown to be sufficiently sensitive to distinguish hemiprotonated folded state and single strand unfolded state of i-motif. The pH of melting has been determined for both sequences in the bulk and at the gold|electrolyte interface. The results evidence a stabilizing effect of the interface on i-motif structure, whereby the pH of melting is higher for the sequences immobilized at the surface. The reversibility and precision of the electrochemical model described here allows a clear and simple characterization of DNA structures and does not require any labeling of the sequence.

Identification of G-quadruplex clusters by high-throughput sequencing of whole-genome amplified products with a G-quadruplex ligand

G-quadruplex (G4) is a DNA secondary structure that has been found to play regulatory roles in the genome. The identification of G4-forming sequences is important to study the specific structure-function relationships of such regions. In the present study, we developed a method for identification of G4 clusters on genomic DNA by high-throughput sequencing of genomic DNA amplified via whole-genome amplification (WGA) in the presence of a G4 ligand. The G4 ligand specifically bound to G4 structures on genomic DNA; thus, DNA polymerase was arrested on the G4 structures stabilised by G4 ligand. We utilised the telomestatin derivative L1H1-7OTD as a G4 ligand and demonstrated that the efficiency of amplification of the G4 cluster regions was lower than that of the non-G4-forming regions. By high-throughput sequencing of the WGA products, 9,651 G4 clusters were identified on human genomic DNA. Among these clusters, 3,766 G4 clusters contained at least one transcriptional start site, suggesting that genes are regulated by G4 clusters rather than by one G4 structure.

Label-Free Detection of Tetramolecular i-Motifs by Surface-Enhanced Raman Spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) can probe DNA primary structure; however, the direct analysis of DNA secondary structure is still technically challenging. In this study, we developed a novel method based on introducing aluminum ion as the aggregation agent for silver nanoparticles, affording high-quality SERS signals of tetramolecular DNA. Thus, the formation of tetramolecular i-motifs was precisely analyzed by SERS signals for the first time and structural features involving glycosidic torsion, protonated cytosine, and cytosine rings were investigated. Moreover, the number of intercalated base pairs in i-motifs were quantified based on the relative intensities of the characteristic SERS bands. The proposed approach would have widespread applications in DNA analysis, especially for detecting various DNA secondary structures.

Spectroscopic investigation on the interaction of DNA with superparamagnetic iron oxide nanoparticles doped with chromene via dopamine as cross linker

Objective(s): The interaction of DNA with iron oxide nanoparticles (SPIONs) was studied to find out the interaction mechanism and design new drug delivery systems. Materials and Methods: The interaction of calf thymus DNA (ctDNA) with SPIONs doped with 2H-chromene via dopamine as cross linker (SPIONs@DA-Chr) was studied using the UV absorption spectroscopy, viscosity measurement, circular dichroism, fluorescence and FT-IR spectroscopic techniques. Results: UV absorption study showed hyperchromic effect in the spectra of DNA. Few changes were observed in the viscosity of ctDNA in the presence of different concentration of SPIONs@DA-Chr. The result of circular dichroism (CD) suggested that SPIONs@DA-Chr can change the secondary structure of DNA. Further, fluorescence quenching reaction of ctDNA with SPIONs@DA-Chr and competitive fluorescence spectroscopy studied by using methylene blue, have shown that the SPIONs@DA-Chr can bind to ctDNA through non-intercalative mode. FT-IR spectroscopy confirmed the binding of SPIONs@DA-Chr and ctDNA. Conclusion: These results suggested that SPIONs@DA-Chr binds to DNA via groove binding mode.

Effect of oligomer length on vibrational coupling and energy relaxation in double-stranded DNA

The effect of oligomer length on the vibrational mode coupling and energy relaxation mechanisms of AT-rich DNA oligomers in double- and single-stranded conformations has been investigated using two-dimensional infrared spectroscopy. Vibrational coupling of modes of the DNA bases to the symmetric stretching vibration of the backbone phosphate group was observed for oligomers long enough to form duplex-DNA structures. The coupling was lost upon melting of the duplex. No significant effect of oligomer length or DNA secondary structure was found on either the timescale for vibrational relaxation of the base modes or the mechanism, which was consistent with a cascade process from base modes to intermediate modes, some of which are located on the deoxyribose group, and subsequently to the phosphate backbone. The study shows that vibrational coupling between base and backbone requires formation of the double-helix structure while vibrational energy management is an inherent property of the nucleotide.

Topoisomerase I and Genome Stability: The Good and the Bad.

Topoisomerase I (Top1) resolves torsional stress that accumulates during transcription, replication and chromatin remodeling by introducing a transient single-strand break in DNA. The cleavage activity of Top1 has opposing roles, either promoting or destabilizing genome integrity depending on the context. Resolution of transcription-associated negative supercoils, for example, prevents pairing of the nascent RNA with the DNA template (R-loops) as well as DNA secondary structure formation. Reduced Top1 levels thus enhance CAG repeat contraction, somatic hypermutation, and class switch recombination. Actively transcribed ribosomal DNA is also destabilized in the absence of Top1, reflecting the importance of Top1 in ensuring efficient transcription. In terms of promoting genome instability, an aborted Top1 catalytic cycle stimulates deletions at short tandem repeats and the enzyme's transesterification activity supports illegitimate recombination. Finally, Top1 incision at ribonucleotides embedded in DNA generates deletions in tandem repeats, and induces gross chromosomal rearrangements and mitotic recombination.

Flanking Variation Influences Rates of Stutter in Simple Repeats.

It has been posited that the longest uninterrupted stretch (LUS) of tandem repeats, as defined by the number of exactly matching repeating motif units, is a better predictor of rates of stutter than the parental allele length (PAL). While there are cases where this hypothesis is likely correct, such as the 9.3 allele in the TH01 locus, there can be situations where it may not apply as well. For example, the PAL may capture flanking indel variations while remaining insensitive to polymorphisms in the repeat, and these haplotypic changes may impact the stutter rate. To address this, rates of stutter were contrasted against the LUS as well as the PAL on different flanking haplotypic backgrounds. This study shows that rates of stutter can vary substantially depending on the flanking haplotype, and while there are cases where the LUS is a better predictor of stutter than the PAL, examples to the contrary are apparent in commonly assayed forensic markers. Further, flanking variation that is 7 bp from the repeat region can impact rates of stutter. These findings suggest that non-proximal effects, such as DNA secondary structure, may be impacting the rates of stutter in common forensic short tandem repeat markers.