DNA Melting Assays Research Articles

DNA Interactions and Biological Activity of 2,9-Disubstituted 1,10-Phenanthroline Thiosemicarbazone-Based Ligands and a 4-Phenylthiazole Derivative.

Four 1,10-phenanthroline derivatives (1-4) were synthesized as potential telomeric DNA binders, three substituted in their chains with thiosemicarbazones (TSCs) and one 4-phenylthiazole derivative. The compounds were characterized using NMR, HRMS, FTIR-spectroscopy and combustion elemental analysis. Quadruplex and dsDNA interactions were preliminarily studied, especially for neutral derivative 1, using FRET-based DNA melting assays, equilibrium dialysis (both competitive and non-competitive), circular dichroism and viscosity titrations. The TSC derivatives bind and stabilize the telomeric Tel22 quadruplex more efficiently than dsDNA, with an estimated 24-fold selectivity determined through equilibrium dialysis for compound 1. In addition, cytotoxic activity against various tumor cells (PC-3, DU145, HeLa, MCF-7 and HT29) and two normal cell lines (HFF-1 and RWPE-1) was evaluated. Except for the 4-phenylthiazole derivative, which was inactive, the compounds showed moderate cytotoxic properties, with the salts displaying lower IC50 values (30-80 μM), compared to the neutral TSC, except in PC-3 cells (IC50 (1) = 18 μM). However, the neutral derivative was the only compound that exhibited a modest selectivity in the case of prostate cells (tumor PC-3 versus healthy RWPE-1). Cell cycle analysis and Annexin V/PI assays revealed that the compounds can produce cell death by apoptosis, an effect that has proven to be similar to that demonstrated by other known 1,10-phenanthroline G4 ligands endowed with antitumor properties, such as PhenDC3 and PhenQE8.

"Dynamical Docking" of Cyclic Dinuclear Au(I) Bis-N-heterocyclic Complexes Facilitates Their Binding to G-Quadruplexes.

With the aim to improve the design of metal complexes as stabilizers of noncanonical DNA secondary structures, namely, G-quadruplexes (G4s), a series of cyclic dinuclear Au(I) N-heterocyclic carbene complexes based on xanthine and benzimidazole ligands has been synthesized and characterized by various methods, including X-ray diffraction. Fluorescence resonance energy transfer (FRET) and CD DNA melting assays unraveled the compounds' stabilization properties toward G4s of different topologies of physiological relevance. Initial structure-activity relationships have been identified and recognize the family of xanthine derivatives as those more selective toward G4s versus duplex DNA. The binding modes and free-energy landscape of the most active xanthine derivative (featuring a propyl linker) with the promoter sequence cKIT1 have been studied by metadynamics. The atomistic simulations evidenced that the Au(I) compound interacts noncovalently with the top G4 tetrad. The theoretical results on the Au(I) complex/DNA Gibbs free energy of binding were experimentally validated by FRET DNA melting assays. The compounds have also been tested for their antiproliferative properties in human cancer cells in vitro, showing generally moderate activity. This study provides further insights into the biological activity of Au(I) organometallics acting via noncovalent interactions and underlines their promise for tunable targeted applications by appropriate chemical modifications.

On the G‐Quadruplex Binding of a New Class of Nickel(II), Copper(II), and Zinc(II) Salphen‐Like Complexes

AbstractThe involvement of non‐canonical DNA structures, such as G‐quadruplex (G4) DNA, in cancer development and progression has set the pace towards the renaissance of DNA‐binding metal complexes. In this work, we report the DNA‐binding of three Ni(II), Cu(II), Zn(II) complexes of a salphen‐like N4‐donor ligand, bearing two imidazole groups condensed with a phenylenediamine moiety. Both duplex and G4 DNAs derived from human telomeres (hTelo), and a sequence mimicking the promoter of the oncogene myc (c‐myc) were studied. UV‐Vis and circular dichroism spectroscopic binding studies pointed out that, while all the three complexes bind the selected oligonucleotides, the Cu(II) derivative is the strongest and G4‐selective compound of the series. Lastly, FRET DNA melting assay results on the Cu(II) complex/hTelo G4 system were interpreted by a loop‐binding mechanism of interaction, as corroborated by molecular dynamics (MD) simulations.

PhenQE8, a Novel Ligand of the Human Telomeric Quadruplex.

A novel quadruplex ligand based on 1,10-phenanthroline and incorporating two guanyl hydrazone functionalities, PhenQE8, is reported herein. Synthetic access was gained in a two-step procedure with an overall yield of 61%. X-ray diffraction studies revealed that PhenQE8 can adopt an extended conformation that may be optimal to favor recognition of quadruplex DNA. DNA interactions with polymorphic G-quadruplex telomeric structures were studied by different techniques, such as Fluorescence resonance energy transfer (FRET) DNA melting assays, circular dichroism and equilibrium dialysis. Our results reveal that the novel ligand PhenQE8 can efficiently recognize the hybrid quadruplex structures of the human telomeric DNA, with high binding affinity and quadruplex/duplex selectivity. Moreover, the compound shows significant cytotoxic activity against a selected panel of cultured tumor cells (PC-3, HeLa and MCF-7), whereas its cytotoxicity is considerably lower in healthy human cells (HFF-1 and RPWE-1).

Exploring the Reactivity and Biological Effects of Heteroleptic N‐Heterocyclic Carbene Gold(I)‐Alkynyl Complexes

With the aim to explore the effects of different organometallic ligands on the reactivity and biological properties of a series of twelve heteroleptic AuI complexes, of general formula [Au(NHC)(alkynyl)] (NHC = benzimidazolylidene or 1,3‐dihydroimidazolylidene) were synthesized and characterized by 1H and 13C NMR and elemental analysis, and in some cases also by X‐ray diffraction. The compounds were all stable in H2O/DMSO as established by NMR spectroscopy, while they could react with model thiols (EtSH) in the presence of water to undergo ligand‐substitution reactions. 1H NMR experiments showed that dissociation of the more labile alkynyl ligand was possible for all compounds, while in the case of the benzimidazolylidene series also dissociation of the NHC ligand could be observed. DFT calculations suggest that, depending on the steric hindrance exerted by both the NHC wingtip groups and the alkynyl substituents, the reaction can proceed either via a π‐stabilized intermediate or with the alkynyl ligand remaining purely σ‐coordinated to the AuI center until completely dissociated. The most stable compounds in PBS buffer (pH 7.4), as assessed by UV‐Visible spectrophotometry, were further investigated for their ability to stabilize G4 DNA by FRET DNA melting assay, showing only moderate activity. Moreover, two derivatives were tested in vitro for their anticancer activities in three different human cancer cell lines and showed cytotoxicity in the low micromolar range.

On the Mechanism of Gold/NHC Compounds Binding to DNA G‐Quadruplexes: Combined Metadynamics and Biophysical Methods

AbstractThe binding modes and free‐energy landscape of two AuI/N‐heterocyclic carbene complexes interacting with G‐quadruplexes, namely a human telomeric (hTelo) and a promoter sequence (C‐KIT1), are studied here for the first time by metadynamics. The theoretical results are validated by FRET DNA melting assays and provide an accurate estimate of the absolute gold complex/DNA binding free energy. This advanced in silico approach is valuable to achieve rational drug design of selective G4 binders.

On the Mechanism of Gold/NHC Compounds Binding to DNA G-Quadruplexes: Combined Metadynamics and Biophysical Methods.

The binding modes and free-energy landscape of two AuI /N-heterocyclic carbene complexes interacting with G-quadruplexes, namely a human telomeric (hTelo) and a promoter sequence (C-KIT1), are studied here for the first time by metadynamics. The theoretical results are validated by FRET DNA melting assays and provide an accurate estimate of the absolute gold complex/DNA binding free energy. This advanced in silico approach is valuable to achieve rational drug design of selective G4 binders.

High Resolution DNA Melting Assays for Detection of Rx1 and Rx2 for High-Throughput Marker-Assisted Selection for Extreme Resistance to Potato virus X in Tetraploid Potato.

Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rx1 and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5Rx1F/5Rx1R primer pair led to reliable detection of Rx1, whereas the 106Rx2F/106Rx2R primer pair detected Rx2 despite some nonspecific reactions in potato clones/cultivars without Rx2. However, the methodology is time consuming and does not differentiate the absence of Rx1/Rx2 from a failed PCR reaction. A newly designed primer pair that targets Rx1 and Rx2 as well as rx1 and rx2 produced an amplicon for all alleles. When the primer pair was combined with 5Rx1F/5Rx1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNA melting (HRM) assay, two distinct melting profiles for Rx1 and rx1, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rx1, Rx2, and rx1/rx2 were also performed. The efficacy of the HRM assays were validated in potato cultivars/clones with known phenotypes, indicating its potential for high-throughput selection of potato clones/cultivars carrying Rx1 or Rx2. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected the presence or absence of Rx1 in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rx1 by HRM exhibited ER to PVX whereas progeny that tested negative for Rx1 were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two Rx1-possessing parents are also discussed.

Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays.

The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective for Isaria javanica and Isaria poprawskii when assayed against all members of the genus. In addition, the high resolution melting profile of this amplicon can be used to discriminate between I. javanica, I. poprawskii and a 1:1 mixture of the two species. The practical application of this technique was confirmed using a bioassay on whitefly nymphs (Bemisia tabaci biotype B) inoculated with I. javanica, I. poprawskii or a 1:1 mixture of the two species. This assay provides a simple assay to identify these two species of entomopathogenic fungi.

Genotyping Sugarcane for the Brown Rust Resistance Locus Bru1 Using Unlabeled Probe Melting

Brown rust, caused by the fungus Puccinia melanocephala, is a major disease of sugarcane (Saccharum spp. hybrid) in Florida, Louisiana, and other sugarcane growing regions. The Bru1 locus has been used as a durable and effective source of resistance, and markers are available to select for the trait. The markers currently being used by the USDA Sugarcane Field Station in Canal Point, FL for Bru1 genotyping have two disadvantages. One marker (here Bru1B) is dominant, which means that a Bru1-negative individual cannot be distinguished from a failed PCR reaction. The second marker (here Bru1A) is codominant, but genotyping requires enzyme restriction and gel electrophoresis, adding time and cost to the analysis. A closed-tube, codominant assay for Bru1 would significantly decrease the time and cost currently required for Bru1 genotyping. By sequencing the Bru1A PCR product from Bru1-positive and Bru1-negative individuals, we indentified two SNPs (here Bru1A1 and Bru1A2) that alter the relevant restriction enzyme recognition sequence. An unlabeled probe assay was designed to target each of the SNPs. Unlabeled probe assays are DNA melting assays that rely on the dissociation of an oligonucleotide probe from its target DNA strand. The dissociation is detected by the decrease in fluorescence of double-stranded DNA binding dyes and is sensitive enough to detect single base pair changes. A comparison between the traditional genotyping method and unlabeled probe melting using 344 genotypes showed a 94.7 % success rate, with 100 % concordance between Bru1A and unlabeled probe genotyping. We also confirmed prior unpublished results that discovered a rare recombination between Bru1A and Bru1B. We have demonstrated that unlabeled probe melting can be used to detect the Bru1 locus, eliminates the risk of false negatives, and is faster and less expensive than the current method.

Abstract 5370: Use of molecular dynamics simulations to rationalise the DNA sequence-selectivity of pyrrolobenzodiazepine-MPB conjugates

Abstract We have previously reported the identification of a GC-interactive non-covalent-binding moiety, a biaryl 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif, through a combined approach involving rational library design and a Fluorescent Intercalation Displacement (FID) assay. This MPB motif, when tethered to a covalently-binding pyrrolobenzodiazepine (PBD) moiety via either an imidazole or pyrrole linkage to form PBD-Imidazole-MPB or PBD-Pyrrole-MPB structures, results in cytotoxicity in the low picomolar range in some tumour cell lines. To assess the DNA-binding affinity and sequence selectivity of PBD-Imidazole-MPB (TSG-1301) and PBD-Pyrrole-MPB (TSG-1302), we have previously reported the use of a FRET-based DNA melting assay based on a duel-labelled hairpin-forming oligonucleotide containing only one PBD binding site. Although having similar cytotoxicities, the sequence selectivity of TSG-1301 and TSG-1302 differ markedly. Melting temperatures (ΔTm) showed that TSG-1301 stabilised both AT- and GC-rich sequences of DNA to an equal extent (i.e., ΔTm of 6°C for each), with TSG-1302 selectively stabilising AT-rich sequences with an approximately 3.5-fold selectivity (i.e., ΔTm of 5°C for AT-rich sequences and 1.4°C for GC-rich). Therefore, Molecular Dynamics (MD) simulations were undertaken to try to understand these differences in sequence selectivity. Simulations were first conducted in implicit solvent for 25ns, and confirmed using extensive explicit solvent simulations using the AMBER software. The results clearly demonstrate the importance of the imidazole ring nitrogen of TSG-1301 in inducing GC-selective interactions through hydrogen bonding. Both the distance between the imidazole ring nitrogen and the terminal ester (an average of 12Å over the duration of simulation), and the shape of the molecule, were found to be crucial for the production of GC interactions, with the Im-MPB moiety ideally spanning side-by-side guanine residues. The AT-selectivity of TSG-1302 could be rationalised using similar techniques, with the Py-MPB moiety contributing to van der Waals interactions between the ligand and AT-rich DNA, due to the MPB moiety presenting a strongly van der Waals-interactive face to the minor-groove. These results were further exemplified using free energy of binding calculations from implicit solvent simulations which provided a difference of -2kcal/mol in favour of the interaction of TSG-1302 with AT-rich compared to GC-rich sequences. The results of this MD study are consistent with observations from our previously reported FRET studies, and the simulations have provided valuable insights into the mode of DNA-interaction of minor groove-binding ligands of this type. This information can be used for the rational design of further generations of agents with improved DNA sequence-selectivity. Citation Format: Paul JM Jackson, Khondaker M. Rahman, David E. Thurston. Use of molecular dynamics simulations to rationalise the DNA sequence-selectivity of pyrrolobenzodiazepine-MPB conjugates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5370. doi:10.1158/1538-7445.AM2014-5370

Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR.

Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests.

Glucose-6-phosphate dehydrogenase (G6PD) gene mutations detection by improved high-resolution DNA melting assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. G6PD gene mutations cause deficiency of the enzyme and a large spectrum of diseases. High-resolution DNA melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. But there was a shortcoming of this method that hemizygous and homozygous genotypes were not easily distinguished from wild-types. Here we used improved HRM method for a small-scale screening of G6PD-deficient variants among people of Meizhou region. Then all amplicons were ascertained by direct DNA sequencing. These results indicated that HRM method was a major technical advance for G6PD mutations screening.

Identification of novel telomeric G-quadruplex-targeting chemical scaffolds through screening of three NCI libraries

Thirteen compounds with diverse chemical structures have been identified as selective telomeric G-quadruplex-binding ligands through screening the NCI Diversity Set II, the NCI Natural Products Set II and the NCI Mechanistic Diversity Set libraries containing a total of 2307 members against a human telomeric G-quadruplex using a FRET-based DNA melting assay. These compounds show significant selectivity towards a telomeric G-quadruplex compared to duplex DNA, fall within a molecular weight range of 327–533, and are generally consistent with the Lipinski Rule of Five for drug-likeness. Thus they provide new chemical scaffolds for the development of novel classes of G-quadruplex-targeting agents.

The prenylated dioxopiperazine alkaloid Cristatin A has selective telomeric DNA G-quadruplex stabilising properties

Cristatin A (1a/b), a prenylated dioxopiperazine alkaloid, has been shown to bind selectively to telomeric quadruplex DNA using a FRET-based DNA melting assay. Crucially, the molecule is more drug-like than most previously identified quadruplex-binding agents, and provides a unique chemical scaffold for future chemical biology and drug discovery studies.

Estimated glomerular filtration rate and its association with the retinol-binding protein 4 (RBP4) locus on human chromosome 10q23

We tested for associations between estimated glomerular filtration rate (eGFR) and retinol-binding protein 4 (RBP4) haplotypes found on human chromosome 10q23. This locus had been linked to eGFR in a previous linkage scan in patients with Type 2 diabetes mellitus. We analysed 469 patients with Type 2 diabetes and 174 normoalbuminuric controls for associations between RBP4 haplotypes and eGFR. For comparison with controls, 295 cases with proteinuria/end-stage renal disease were tested for associations with advanced diabetic nephropathy. Genotyping was performed using high-resolution DNA melting assays. Data analysis was performed using the haplo.stats package. Genetic variations in RBP4 were not associated with advanced diabetic nephropathy. Compared with the common A/G/G/C haplotype, C/A/A/C carriers among the normoalbuminuric controls had higher eGFR values among younger patients but lower eGFRs among the older patients (effect size=2.2, P=3.3×10(-7)). Furthermore, while eGFR values were fairly consistent over the range of systolic blood pressure (SBP) values for the common haplotype, eGFR in C/A/A/C carriers increased with SBP (effect size=3.6, P=1.5×10(-2)). There was a significant interaction between the C/A/A/C haplotype and HbA1c as they affect eGFR compared to the common haplotype (effect size=2.1, P=2.1×10(-3)). Power calculations demonstrated that our study had >90% power to detect the observed interactions even while performing multiple hypotheses testing. The interaction between SBP and the C/A/A/C haplotype remained significant (P=2.8×10(-2)) even when these three haplotype-environment interactions were simultaneously estimated. RBP4 haplotypes may be important in genetically modulating renal function in response to environmental challenges among patients with Type 2 diabetes.

Rapid Molecular Analysis of the STAT3 Gene in Job Syndrome of Hyper-IgE and Recurrent Infectious Diseases

With the recent discovery of mutations in the STAT3 gene in the majority of patients with classic Hyper-IgE syndrome, it is now possible to make a molecular diagnosis in most of these cases. We have developed a PCR-based high-resolution DNA-melting assay to scan selected exons of the STAT3 gene for mutations responsible for Hyper-IgE syndrome, which is then followed by targeted sequencing. We scanned for mutations in 10 unrelated pedigrees, which include 16 patients with classic Hyper-IgE syndrome. These pedigrees include both sporadic and familial cases and their relatives, and we have found STAT3 mutations in all affected individuals. High-resolution melting analysis allows a single day turn-around time for mutation scanning and targeted sequencing of the STAT3 gene, which will greatly facilitate the rapid diagnosis of the Hyper-IgE syndrome, allowing prompt and appropriate therapy, prophylaxis, improved clinical outcome, and accurate genetic counseling.

DNA Melting in Gold Nanostove Clusters

Clusters of gold nanoparticles can be used as optically driven stoves to locally heat biological material such as DNA in biosensors or cancerous tissue in thermotherapy. Clusters of gold nanoparticles behave distinctively different from single nanoheaters as they accumulate heat in-between the individual nanoparticles. However, in very large clusters the rear nanoparticles are shed by front-lying nanoparticles such that the optothermal heating saturates with increasing cluster size. We focus on these two effects and describe the temporal and spatial development of heat generation and subsequent cooling of nanoparticle clusters excited by nanosecond laser pulses. It is found that (i) heat accumulation is most important for DNA melting assays to generate enough heat for DNA melting, (ii) shadowing effects are significant in micrometer sized nanoparticle clusters, and (iii) that DNA needs to be heated substantially above the equilibrium thermodynamic melting temperature in order to induce DNA melting by nanosecond laser pulses.

Gold NanoStoves for Microsecond DNA Melting Analysis

In traditional DNA melting assays, the temperature of the DNA-containing solution is slowly ramped up. In contrast, we use 300 ns laser pulses to rapidly heat DNA bound gold nanoparticle aggregates. We show that double-stranded DNA melts on a microsecond time scale that leads to a disintegration of the gold nanoparticle aggregates on a millisecond time scale. A perfectly matching and a point-mutated DNA sequence can be clearly distinguished in less than one millisecond even in a 1:1 mixture of both targets.

Genotyping HIV-1 and HCV Strains by a Combinatorial DNA Melting Assay (COMA)

Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) strains can be genetically classified into genetic lineages known as genetic types or subtypes according to phylogenetic analyses of complete or partial nucleotide sequences of their genomes. The genetic classification of HIV-1 and HCV strains has important implications for the development of globally effective vaccines and for the management of patients. A new method, termed combinatorial DNA melting assay (COMA), allows rapid accessing of comparative genetic information between related DNA sequences, making it possible to rapidly and accurately genotype unknown HIV-1 and HCV strains. COMA is mainly based on the differential melting properties of long DNA heteroduplexes. Combinatorial arrays of DNA heteroduplexes are formed when captured PCR-amplified reference DNA with known nucleotide sequences are combined with solution-phase complementary and antigenically labeled DNA with unknown sequences. Genetic divergence between the known and the unknown sequences is inferred as the experimentally derived melting curves of the two strands of the DNA heteroduplexes increasingly diverge. COMA was successfully applied to the genetic classification of HIV-1 and HCV strains into phylogenetic lineages or subtypes. Use of this assay should accelerate current efforts to understand the global molecular epidemiology of HIV-1 and HCV and may extend to the genetic characterization of other genetically diverse infectious pathogens associated with numerous diseases.