Rich DNA Sequences Research Articles

Differential binding of curcumin with chair and basket type anti-parallel DNA G-quadruplexes

Guanine rich DNA sequences are widespread throughout the genome and they are prone to form G-quadruplex structures both in-vitro and in-vivo. Owing to their regulatory roles in important biological reactions, DNA G-quadruplexes are the prime target for the development of anti-cancer drug molecules. In the present study, we have investigated the interaction of curcumin with anti-parallel chair and basket type DNA G-quadruplexes formed by thrombin binding aptamer (TBA) and human telomeric repeat (HTG) sequences, respectively. The results obtained from spectroscopic techniques demonstrated that curcumin interacts with both the DNA G-quadruplexes through external binding mode and the estimated apparent binding constant value (Kb) was slightly higher for HTG (Kb = (4.38 ± 0.09) × 105 M−1) than that estimated for TBA (Kb = (0.62 ± 0.02) × 105 M−1) DNA G-quadruplex structures. It was also revealed that upon binding with curcumin, TBA and HTG DNA G-quadruplexes were slightly stabilized and destabilized, respectively. Circular dichroism study revealed that upon binding with curcumin the global structure of both the DNA G-quadruplexes was unaltered, whereas the local structure of HTG DNA G-quaruplex was slightly altered. Conjointly all the aforementioned spectroscopic methods emphasize that curcumin has differential interaction with anti-parallel DNA G-quadruplex structures depending on their loop orientation.

Organic-soluble cytosine derivatives for studying base-pair interactions in nonaqueous solution

The presence of higher order G-quadruplex-DNA (G4) structures in human cells in guanine-rich DNA sequences has been well established. In cases of G4 helicase impairment or G4 stabilization via association with G4 ligands, it has been determined that arrest and/or termination of DNA transcription/replication occurs because these structures act as physical barriers to both helicase and polymerase mobility along the DNA. Truncation of transcription or replication may result in the loss of genetic material expression leading to disorders. To ameliorate G4-induced barriers, agents that destabilize or “unwind” the quadraplex structures are sought. Earlier work has shown that the cytosine analogue phenylpyrrolocytosine has potential G4-destabilizing ability. It was hypothesized that the unwinding activity was related to its ability to base pair with guanosine. To facilitate the design and evaluation of the hydrogen bonding ability of cytosine analogues, solubility in aprotic, and low-polarity solvents, e.g., deutero-chloroform (CDCl3), is needed; then, determination of association constants ( Ka) with guanine via 1H nuclear magnetic resonance (NMR) spectroscopy or isothermal titration calorimetry (ITC) will be possible. Herein, we report on the synthesis of three new chloroform-soluble, cytosine analogues: N1-cyclohexylmethyl-7-phenylimidazolocytosine, N1-cyclohexylmethyl-7-(6-(methoxycarbonyl)pyridin-2-yl)imidazolocytosine, and N1-cyclohexylmethyl-7-(6-carbamoylpyridin-2-yl)imidazolocytosine. We further report the binding interactions of each of these analogues with the guanosine derivative 2′,3′,5′- O-tris( tert-butyldimethylsilyl)guanosine through the use of both ITC and 1H NMR titration experiments in chloroform and compare the binding to persilyated ribo-cytidine.

Crol contributes to PRE-mediated repression and Polycomb group proteins recruitment in Drosophila.

The Polycomb group (PcG) proteins are fundamental epigenetic regulators that control the repressive state of target genes in multicellular organisms. One of the open questions is defining the mechanisms of PcG recruitment to chromatin. In Drosophila, the crucial role in PcG recruitment is thought to belong to DNA-binding proteins associated with Polycomb response elements (PREs). However, current data suggests that not all PRE-binding factors have been identified. Here, we report the identification of the transcription factor Crooked legs (Crol) as a novel PcG recruiter. Crol is a C2H2-type Zinc Finger protein that directly binds to poly(G)-rich DNA sequences. Mutation of Crol binding sites as well as crol CRISPR/Cas9 knockout diminish the repressive activity of PREs in transgenes. Like other PRE-DNA binding proteins, Crol co-localizes with PcG proteins inside and outside of H3K27me3 domains. Crol knockout impairs the recruitment of the PRC1 subunit Polyhomeotic and the PRE-binding protein Combgap at a subset of sites. The decreased binding of PcG proteins is accompanied by dysregulated transcription of target genes. Overall, our study identified Crol as a new important player in PcG recruitment and epigenetic regulation.

Interaction of isoquinoline alkaloids with pyrimidine motif triplex DNA by mass spectrometry and spectroscopies reveals diverse mechanisms

Isoquinoline alkaloids represent an important class of molecules due to their broad range of pharmacology and clinical utility. Prospective development and use of these alkaloids as effective anticancer agents have elicited great interest. In this study, in order to reveal structure-activity relationship, we present the characterization of bioactive isoquinoline alkaloid-DNA triplex interactions, with particular emphasis on the sequence selectivity and preference of binding to the two types of DNA triplexes, by electrospray ionization mass spectrometry (ESI-MS) and various spectroscopic techniques. The six alkaloids, including coptisine, columbamine, epiberberine, berberrubine, jateorhizine, and fangchinoline, were selected to explore their interactions with the TC and TTT triplex DNA structures. Berberrubine, fangchinoline, coptisine, columbamine, and epiberberine have preference for TC rich DNA sequences compared to TTT rich DNA triplex based on affinity values in MS. The experimental results from different fragmentation modes in tandem MS, subtractive and hyperchromic effects in UV absorption spectra, fluorescence quenching and enhancement in fluorescence spectra, and strong conformational changes in circular dichroism (CD) hinted that the interaction between isoquinoline alkaloid-TC/TTT DNA had diverse mechanisms including at least two different binding modes: the electrostatic binding and the intercalation binding. Interestingly, columbamine, berberrubine, and fangchinoline can stabilize TTT triplex as inferred from optical thermal melting profiles, while it was not the case in TC triplex. These results provide new insights into binding of isoquinoline alkaloids to pyrimidine motif triplex DNA.

Third-generation sequencing: A novel tool detects complex variants in the α-thalassemia gene

ObjectiveThalassemia is a monogenic disorder with a high carrier rate in the southern region of China. Most laboratories currently follow the protocol of testing hematologic indicators in individuals with positive hematologic indicators and then using the hot-spot mutation test kit. A novel thalassemia gene test is performed if there is a mismatch between the hematology and hot-spot mutation test results. However, due to the large population in southern China, some individuals carry complex α-globin gene cluster (CAGC) variants in NG_000006.1, which are difficult to detect using conventional thalassemia genetic analysis protocols, leading to missed or false genetic test results for individuals carrying these complex α-globin gene cluster variants. When an individual carries a complex α-thalassemia gene variant, and an individual carries a β- thalassemia gene variant, there may be clinical symptoms that might complicate clinical consultation and prenatal diagnosis if not accurately detected. Third-generation sequencing (TGS) enables long-read single-molecule sequencing with high detection accuracy, and long-length DNA chain reads in high-fidelity reads mode. TGS can be used to analyze high homology and rich GC DNA sequences. ResultsFour samples that showed abnormalities in the thalassemia genetic test were studied using TGS, revealing that they carried genotypes with complex α-globin gene cluster variants, one of which was a complex variant αα anti3.7 α anti3.7 α 17.2. ConclusionsTGS detects complex α-globin gene cluster variants. This study may provide a reference protocol for the use of TGS for the detection of complex α-globin gene cluster variants. TGS can reveal individuals with complex α-thalassemia genotypes in the population and improve the accuracy of genetic counseling and prenatal diagnosis.

Oncofetal genes and its association with bivalent chromatin and DNA methylation: (A Review)

Oncofetal genes are primarily those genes that are expressed in fetal tissue but remain in a state of inactivation in adult. Assessing the various factors involved in reactivation of oncofetal gene, the predominant one that represent a distinct mechanism is bivalency. Evidence suggest that DNA methylation in context to bivalent domain of Histone 3 particularly at Lysine 4 and 27, serves as a major site for reactivation of silenced transcriptional activity along with protein complexes Tri-thorax (TrxG) and Poly-comb(PcG). The malignancy of proto- oncogene establishes a correlation with CpG rich DNA sequence that adds appropriate methylation providing rapid activation to express the silenced genes.

G-Quadruplexes Formation at the Upstream Region of Replication Origin (OriL) of the Pseudorabies Virus: Implications for Antiviral Targets

Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses’ genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV’s attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.

Multiple G-quadruplex binding ligand induced transcriptomic map of cancer cell lines.

The G-quadruplexes (G4s) are a class of DNA secondary structureswith guanine rich DNA sequences that can fold into four stranded non-canonical structures. At the genomic level, their pivotal role is well established in DNA replication, telomerase functions, constitution of topologically associating domains, and the regulation of gene expression. Genome instability mediated by altered G4 formation and assembly has been associated with multiple disorders including cancers and neurodegenerative disorders. Multiple tools have also been developed to predict the potential G4 regions in genomes and the whole genome G4 maps are also being derived through sequencing approaches. Enrichment of G4s in the cis-regulatory elements of genes associated with tumorigenesis has accelerated the quest for identification of G4-DNAbinding ligands (G4DBLs) that can selectively bind and regulate the expression of such specific genes. In this context, the analysis of G4DBL responsive transcriptome in diverse cancer cell lines is inevitable for assessment of the specificity of novel G4DBLs. Towards this, we assembled the transcripts differentially regulated by different G4DBLs and have also identified a core set of genes regulated in diverse cancer cell lines in response to 3 or more of these ligands. With the mode of action of G4DBLs towards topology shifts, folding, or disruption of G4 structure being currently visualized, we believe that this dataset will serve as a platform for assembly of G4DBL responsive transcriptome for comparative analysis of G4DBLs in multiple cancer cells based on the expression of specific cis-regulatory G4 associated genes in the future.

The advancement of telomere quantification methods.

Telomeres, guanine rich DNA sequences, which are found at both ends of human chromosomes, play a vital role in genome protection. These repetitive nucleotide sequences protect the genome from nucleolytic degradation, unnecessary recombination, and interchromosomal fusion. Though, as somatic cells go through replication cycles, their telomeres shrink until they reach a critical length called the Hayflick limit. At this limit, cellular senescence, an irreversible cell cycle arrest, is prompted. For all the above reasons, telomere length is a hopeful biomarker for age-associated diseases and cancer. While there are numerous methods for telomere measurement and quantification, there are still challenges for routine analysis in clinics as these methods are not simple and rapid. Recently, a new method has been developed that measures absolute length and absolute quantities of single telomere molecules. This method, single telomere absolute-length rapid (STAR) assay, which promises to measure telomere length rapidly and accurately, is also expected to be scalable. This review will discuss different telomere length measurement methods, including STAR assay, and will highlight each of their advantages and drawbacks. It will culminate in determining if STAR assay has the potential to be the superior method for telomere measurement.

Indel-K2P: a modified Kimura 2 Parameters (K2P) model to incorporate insertion and deletion (Indel) information in phylogenetic analysis

ABSTRACT Evolutionary models are widely used to estimate the evolutionary relationship between the various biological taxa . Majority of the existing evolutionary models ignore insertion and deletion (indel) event, which reduces the computational difficulties, but at the same time, it causes loss of valuable phylogenetic information. In this study, we have developed a modified Kimura 2 parameters (K2P) model to utilise the indel information in phylogenetic tree construction especially in case of low substitution rate sequences Indel rich highly similar DNA sequences (trnL-trnF intergenic spacer region) of Phalaenopsis were collected from Genbank and to test the efficiency of the model in phylogenetic. Our developed Indel-K2P model and K2P model were implemented in MATLAB and calculated the bootstrap value and Robinson-Foulds (RF) distance to test the confidence and accuracy of phylogenetic tree topology. The phylogenetic trees constructed within the framework of Indel-K2P model, exhibited 92.66% accuracy and 40.9% root node confidence while the K2P model without indel shows less accuracy and less root node confidence. Our results indicate that modified Kimura 2 parameters with indel (Indel-K2P) model performed better than Kimura 2 Parameters (K2P) in phylogenetic tree construction for indel rich DNA sequences.

Determination of the binding of natural products to the human c-myb oncogene promoter G-quadruplex DNA by capillary electrophoresis and electrospray ionization mass spectrometry

The relationship between a drug and its target directly affects its pharmacology and efficacy. Drug-target binding ability and binding stoichiometry are essential characterization data in pharmaceutical research. The c-myb proto-oncogene encodes a crucial transcription factor that is involved in proliferation, differentiation, and maturation during hematopoiesis. Recent studies have found that the human oncogene c-myb is overexpressed in cancer tissues such as colorectal cancer. C-myb has become a potential therapeutic target for colorectal cancer, leukemia, and other cancers. A guanine (G)-rich DNA sequence located in the promoter region of c-myb can be spontaneously folded to form an intra-molecular G-quadruplex (G4) with cationic induction. The specific recognition of small molecules can stabilize this G4 folding, thus regulating the transcription and expression of c-myb. In this study, pressure assisted capillary electrophoresis frontier analysis (PACE-FA) combined with electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interactions between the human c-myb promoter G4 and natural product molecules. In PACE-FA, an external pressure (no more than 13.8 kPa) in the same direction of the migration of the analyte was used in capillary electrophoresis frontier analysis (CE-FA), which greatly sped up the analysis while maintaining the accuracy of the results. Meanwhile, the combination of PACE-FA and ESI-MS could rapidly determine the affinity and stoichiometric relationship between binding molecules and targets. First, the intramolecular parallel-stranded G4 formation of the c-myb promoter sequence in the presence of cations (K+, NH4+) was investigated by circular dichroism (CD) and ESI-MS. Then, ESI-MS was used to rapidly screen the natural products for candidate molecules that bound the target G4 DNA. The binding interactions were measured by mixing the c-myb DNA with each natural product separately in a 1:4 molar ratio, and then directly infusing these mixtures into the ESI-MS system. From the ESI-MS spectra, IRa values calculated from the relative intensities of DNA and its complex ions were used to probe the binding affinities of the natural products. This parameter denotes the relative binding affinity for a small molecule with the G4 DNA. Three natural products were identified through the screen, and their binding affinities to G4 DNA were ranked as follows:pseudolaric acid B > scopolamine butylbromide > nuciferine. Considering that both specific and non-specific binding existed in the solution phase, a free solution method using PACE-FA was developed to further test the binding ability of these products to the c-myb promoter G4 DNA. In the PACE-FA experiments, the pre-equilibrated mixture of the c-myb G4 DNA and the selected ligand was injected into the capillary prior to separation. Upon applying voltage and an external pressure (6.9 kPa) to the capillary, different species of analytes in the sample migrated at their own rates due to their different sizes and charges. The results showed that scopolamine butylbromide could bind specifically to target G4 DNA with 1:1 stoichiometry and a binding constant of 1.18×105 L/mol. Nuciferine's binding to G4 DNA showed a linear increasing trend due to nonspecific binding; thus nuciferine is a nonspecific binder. Although pseudolaric acid B showed high affinity for the c-myb G4 DNA and 1:1 and 1:2 G4-bound complex ions were observed in ESI-MS measurements, the PACE-FA results indicated that pseudolaric acid B did not bind to target G4 DNA in free solution. Therefore, scopolamine butylbromide could be the best candidate to regulate the transcription of the c-myb oncogene, and is expected to be a precursor for anticancer drugs. In this work, PACE-FA has allowed for significant improvements to the conventional CE-FA technique. This combination of PACE-FA and ESI-MS not only reduced the time needed for binding analysis, but also improved the accuracy and specificity of the affinity analysis compared to conventional binding approaches. Furthermore, this combination could be used to screen other targeted drug candidates and to evaluate their interaction mechanisms.

Slow motions in A\xb7T rich DNA sequence

In free B-DNA, slow (microsecond-to-millisecond) motions that involve equilibrium between Watson–Crick (WC) and Hoogsteen (HG) base-pairing expand the DNA dynamic repertoire that could mediate DNA–protein assemblies. R1ρ relaxation dispersion NMR methods are powerful tools to capture such slow conformational exchanges in solution using 13C/15 N labelled DNA. Here, these approaches were applied to a dodecamer containing a TTAAA element that was assumed to facilitate nucleosome formation. NMR data and inferred exchange parameters assign HG base pairs as the minor, transient conformers specifically observed in three successive A·T base pairs forming the TAA·TTA segment. The abundance of these HG A·T base pairs can be up to 1.2% which is high compared to what has previously been observed. Data analyses support a scenario in which the three adenines undergo non-simultaneous motions despite their spatial proximity, thus optimising the probability of having one HG base pair in the TAA·TTA segment. Finally, revisiting previous NMR data on H2 resonance linewidths on the basis of our results promotes the idea of there being a special propensity of A·T base pairs in TAA·TTA tracts to adopt HG pairing. In summary, this study provides an example of a DNA functional element submitted to slow conformational exchange. More generally, it strengthens the importance of the role of the DNA sequence in modulating its dynamics, over a nano- to milli-second time scale.

Prediction of Hit-to-Lead Ligand Molecule Interaction with G-Quadruplex DNA from c-Myc Oncogene Promoter Region

Targeting guanine (G)-rich DNA sequences, folded into non-canonical G-quadruplex (G4) structures, by small ligand molecules is a potential strategy for gene therapy of cancer disease. BRACO-19 has been recently established as a unique (thermodynamically favorable and highly selective) binder, being involved in the external stacking mode of interaction with a G4-DNA formed in the c-Myc oncogene promoter region (P. M. Mitrasinovic, Croat. Chem. Acta 2019, 92, 43-57). Herein, hit-to-lead ligands are identified using high-throughput virtual screening (HTVS). Search of the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases is performed using the key pharmacophore features of BRACO-19. At the very outset, out of a total of 29,009 entries, 95 hits are extracted and evaluated by docking them in the binding sites of G4. Then, 22 hits are chosen by observing the binding free energies. Consequently, 3 hit-to-lead candidates are selected on the basis of structural criteria. Finally, a lead candidate structure is proposed using analog design and considering both the physicochemical requirements for optimal biological activity and a variety of pharmacological standpoints. Implications of the present study for experimental research are discussed.

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes.

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

Revealing unfolding steps and volume changes of human telomeric i-motif DNA.

Cytosine rich DNA sequences can fold into a non-canonical four stranded intercalated i-motif structure. We investigated Htel-iM, an i-motif structure of the human telomeric DNA region that plays an important role in cell division, cancer diseases and aging. A high pressure up to 1 Gpa was applied to reveal the volumetric changes during unfolding. Thermal transitions at different pressures were followed by infrared and fluorescence spectroscopy. We demonstrated that Htel-iM unfolds in two steps. First the outer hydrogen bonds break in which C(2)[double bond, length as m-dash]O(2) and the N(4)H2 amino group is involved, subsequently the hydrogen bond involving N(3)+ becomes broken. Htel-iM was destabilized by pressure and unfolded with a negative volume change.

Self-crowding influences the temperature – pressure stability of the human telomere G-quadruplex

We measured the effect of crowded environment on G-quadruplex structures, formed by guanine rich DNA sequences. Fluorescence and infrared spectroscopy were used to determine the temperature stability of G-quadruplex structure formed by the human telomere sequence. We determined the T-p phase diagram of Htel aptamer up to 1 GPa at different self-crowding conditions. The unfolding volume change was determined from the pressure induced shift of the unfolding temperature of the quadruplex form. The unfolding volume change decreased in magnitude, and even its sign changed from negative (−19 ml/mol) to positive (7 ml/mol) under self-crowded conditions. The possible explanations are the appearance of the parallel GQ structure at high concentration or the fact that the volume decrease caused by the released central K+ ion during the unfolding is less significant in crowded environment.

The four repeat Giardia lamblia telomere forms tetramolecular G-quadruplex with antiparallel topology

Guanine rich DNA sequences of regulatory genomic regions form secondary structures known as G-quadruplexes usually stabilized by tetrads of Hoogsteen hydrogen bonded guanines. The in vivo existence of G-quadruplexes ascertains their biological roles. Human telomeric repeats are the most studied G-rich sequences. The four repeat Giardia telomeric sequence (TAGGG)4 differs from its human counterpart (TTAGGG)4, by deletion of one T at the G-tract intervening site of each repeat. We show here that whilst the two repeat Giardia telomeric sequence (TAGGG)2 forms parallel and antiparallel quadruplexes with tetramolecular topology exclusively, the four repeat version (TAGGG)4 forms a tetramolecular (antiparallel) and unimolecular (parallel) quadruplexes in Na+. The tetramolecular (antiparallel) G-quadruplex formed by four repeats of Giardia telomeric sequence is stabilized by the additional Watson-Crick bonding between its intervening TA bases aligned in antiparallel fashion. Four stranded antiparallel quadruplex for four repeats of any telomeric sequence have not been characterized till date. We hypothesize that telomeric association in antiparallel fashion, (via G-overhangs to form tetramolecular quadruplex) could be a biologically relevant molecular event. Further, coexistence of Hoogsteen as well as Watson-Crick base pairing might give insight for recognition of conformationally diverse DNA structures by ligands. Communicated by Ramaswamy H. Sarma

Towards revealing the molecular mechanism of STF

Leaf is a primary organ for the plants to sustain their life and support life on earth by assimilating solar energy and carbon dioxide into chemical energy. STENOFOLIA (STF), a WUSCHEL (WUS)‐like homeobox transcriptional regulator, plays a key role in embryonic patterning, blade outgrowth, and leaf vascular patterning. STF has also been shown to help in increasing biomass and solubilized sugar yield. STF does not affect leaf outgrowth directly but is critical for their development. It is known that transcriptional repressor activity mediated by the WUSbox of STF acts to promote blade outgrowth, and STF executes its functions with its interactions with other protein and DNA. STF binds to GA rich DNA sequence and there is specific DNA binding domain present in the sequence. Although the DNA binding domain (HD) of the protein is conserved across the species, the WOX family HD seems to be unique and cannot be replaced by another HD protein from the plant. WOX HD domain is longer than usual HD domains and has additional protein interaction sites to achieve stable repressive activity. The molecular mechanism of STF function and how WOX genes have adopted the WUS in evolutionary development are unknown. Here we are working towards determining the atomic structure of STF in complex with DNA by using x‐ray crystallography.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Binding of anticancer drug daunomycin to parallel G-quadruplex DNA [d-(TTGGGGT)]4 leads to thermal stabilization: A multispectroscopic investigation

Guanine rich DNA sequences form four stranded G-quadruplex structures found in telomeric DNA and oncogene promoters. Small molecules binding and stabilizing the G-quadruplex disrupt telomere maintenance and gene regulation, thereby limiting the proliferation of cancer cells. The anti-cancer drug daunomycin binds to both G-quadruplex DNA and duplex DNA. We have undertaken a study of interaction of daunomycin to [d-(TTGGGGT)]4 comprising telomeric DNA sequence from Tetrahymena thermophilia to understand the mechanisms of its action. Absorbance, fluorescence and circular dicroism spectra show significant change on interaction with no change in wavelength maxima. The daunomycin dimers present in free state in solution are disrupted on binding. Presence of all sequential short inter proton distance contacts in nuclear magnetic resonance spectra confirm external binding. The observed inter molecular nuclear Overhauser enhancements, changes in chemical shift and molecular docking studies establish well defined binding of daunomycin at two different sites of G-quadruplex DNA. Thermal stabilization of [d-(TTGGGGT)]4 by 10–15 °C upon daunomycin binding is expected to reduce access of telomerase to its functional site at telomeres. The present studies on mode of action pave the way for alternate derivatives/analogues by chemical modification of anthracyclines to arrive at a more potent telomerase inhibitor.

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.