Computational Assessment of a Dual-Action Ru(II)-Based Complex: Photosensitizer in Photodynamic Therapy and Intercalating Agent for Inducing DNA Damage.

A combined quantum-mechanical and classical molecular dynamics study of a recent Ru(II) complex with potential dual anticancer action is reported here. The main basis for the multiple action relies on the merocyanine ligand, whose electronic structure allows the drug to be able to absorb within the therapeutic window and in turn efficiently generate 1O2 for photodynamic therapy application and to intercalate within two nucleobases couples establishing reversible electrostatic interactions with DNA. TDDFT outcomes, which include the absorption spectrum, triplet states energy, and spin-orbit matrix elements, evidence that the photosensitizing activity is ensured by an MLCT state at around 660 nm, involving the merocyanine-based ligand, and by an efficient ISC from such state to triplet states with different characters. On the other hand, the MD exploration of all the possible intercalation sites within the dodecamer B-DNA evidences the ability of the complex to establish several electrostatic interactions with the nucleobases, thus potentially inducing DNA damage, though the simulation of the absorption spectra for models extracted by each MD trajectory shows that the photosensitizing properties of the complex remain unaltered. The computational results support that the anti-tumor effect may be related to multiple mechanisms of action.

Colorimetric loop-mediated isothermal amplification (LAMP) for cost-effective and quantitative detection of SARS-CoV-2: the change in color in LAMP-based assays quantitatively correlates with viral copy number.

We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).

Molecular methods for assessment of non-covalent metallodrug-DNA interactions.

The binding of small molecule metallodrugs to discrete regions of nucleic acids is an important branch of medicinal chemistry and the nature of these interactions, allied with sequence selectivity, forms part of the backbone of modern medicinal inorganic chemistry research. In this tutorial review we describe a range of molecular methods currently employed within our laboratories to explore novel metallodrug-DNA interactions. At the outset, an introduction to DNA from a structural perspective is provided along with descriptions of non-covalent DNA recognition focusing on intercalation, insertion, and phosphate binding. Molecular methods, described from a non-expert perspective, to identify non-covalent and pre-associative nucleic acid recognition are then demonstrated using a variety of techniques including direct (non-optical) and indirect (optical) methods. Direct methods include: X-ray crystallography; NMR spectroscopy; mass spectrometry; and viscosity while indirect approaches detail: competitive inhibition experiments; fluorescence and absorbance spectroscopy; circular dichroism; and electrophoresis-based techniques. For each method described we provide an overview of the technique, a detailed examination of results obtained and relevant follow-on of advanced biophysical/analytical techniques. To achieve this, a selection of relevant copper(ii) and platinum(ii) complexes developed within our laboratories are discussed and are compared, where possible, to classical DNA binding agents. Applying these molecular methods enables us to determine structure-activity factors important to rational metallodrug design. In many cases, combinations of molecular methods are required to comprehensively elucidate new metallodrug-DNA interactions and, from a drug discovery perspective, coupling this data with cellular responses helps to inform understanding of how metallodrug-DNA binding interactions manifest cytotoxic action.

Bisintercalating DNA redox reporters for real-time electrochemical qLAMP.

The electrochemical detection methods have emerged as a potential alternative to the bench-top optical systems in monitoring nucleic acid amplification. DNA intercalating redox reporters play a crucial role in such monitoring schemes. Here, a series of bisintercalating redox probes have been tailor-made to meet specific requirements of electrochemical quantitative loop-mediated isothermal amplification (qLAMP). The probes composed of two naphthoquinone-imidazole (NQIM) derivatives as signal motifs that are covalently bridged by different linkers (R). They are bis-NQIM-R; R = Alkane (Ak), ethylene glycol (EG) and phenyl (Ph). The linkers allow the probes to be fine-tuned for securing ideal redox reporter. DNA binding studies via electrochemical and fluorescence techniques demonstrate that the bis-NQIM-R probes possess better ds-DNA bisintercalating ability compared to their mono-analogs. The bis-NQIM-Ph was implemented in a real-time electrochemical qLAMP, for which a prototype custom-made device that can perform fully automated multiplexed analyses is devised. A single copy of Salmonella DNA was quantified in just 10 min and the performance is comparable to the benchtop fluorescence method. Thus, the bisintercalating redox reporters incorporated electrochemical detection schemes hold great promise in qLAMP.

Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/µL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

Assessment of SYBR green I dye-based fluorescence assay for screening antimalarial activity of cationic peptides and DNA intercalating agents.

The SYBR green I (SG) dye-based fluorescence assay for screening antimalarial compounds is based on direct quantitation of parasite DNA. We show that DNA-interacting cationic cell-penetrating peptides (CPPs) and intercalating agents compete with SG dye to bind to DNA. Therefore, readouts of this assay, unlike those of the [(3)H]hypoxanthine incorporation assay, for the antimalarial activity of the above DNA binding agents may be erroneous. In the case of CPPs, false readouts can be improved by the removal of excess peptides.

Effect of knots on binding of intercalators to DNA.

We study the effect of knots in circular dsDNA molecules on the binding of intercalating ligands. Using Monte Carlo simulations we show that depending on their handedness, the presence of knots can either suppress or enhance intercalation in supercoiled DNA. When the occupancy of intercalators on DNA is low, the effect of knots on intercalation can be captured by introducing a shift in the mean writhe of the chain that accounts for the writhe of the corresponding ideal knot. In the limit of high intercalator occupancy, the writhe distribution of different knots is strongly affected by excluded volume effects and therefore by salt concentration. Based on the finding that different knots yield well-separated probability distributions of bound intercalators, we propose a new experimental approach to determine DNA topology by monitoring the intensity of fluorescence emitted by dye molecules intercalated into knotted DNA molecules.

Role of intercalation and redox potential in DNA photosensitization by ruthenium(II) polypyridyl complexes: assessment using DNA repair protein tests.

Here we report that the photoreactivity of ruthenium(II) complexes with nucleobases may not only be modulated by their photoredox properties but also by their DNA binding mode. The damage resulting from photolysis of synthetic oligonucleotides and plasmid DNA by [Ru(bpz)3](2+), [Ru(bipy)3](2+) and the two DNA intercalating agents [Ru(bpz)2dppz](2+) and [Ru(bipy)2dppz](2+) has been monitored by polyacrylamide gel electrophoresis and by tests using proteins involved in DNA repair processes (DNA-PKCs, Ku80, Ku70, and PARP-1). The data show that intercalation controls the nature of the DNA damage photo-induced by ruthenium(II) complexes reacting with DNA via an electron transfer process. The intercalating agent [Ru(bpz)2dppz](2+) is a powerful DNA breaker inducing the formation of both single and double (DSBs) strand breaks which are recognized by the PARP-1 and DNA-PKCs proteins respectively. [Ru(bpz)2dppz](2+) is the first ruthenium(II) complex described in the literature that is able to induce DSBs by an electron transfer process. In contrast, its non-intercalating parent compound, [Ru(bpz)3](2+), is mostly an efficient DNA alkylating agent. Photoadducts are recognized by the proteins Ku70 and Ku80 as with cisplatin adducts. This result suggests that photoaddition of [Ru(bpz)2dppz](2+) is strongly affected by its DNA intercalation whereas its photonuclease activity is exalted. The data clearly show that DNA intercalation decreases drastically the photonuclease activity of ruthenium(II) complexes oxidizing guanine via the production of singlet oxygen. Interestingly, the DNA sequencing data revealed that the ligand dipyridophenazine exhibits on single-stranded oligonucleotides a preference for the 5'-TGCGT-3' sequence. Moreover the use of proteins involved in DNA repair processes to detect DNA damage was a powerful tool to examine the photoreactivity of ruthenium(II) complexes with nucleic acids.

[Assessment of the distribution of nucleic acid intercalators in yeast cells by the pseudospectral image analysis].

The intracellular location of nucleic acid intercalators (NAI) in live (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: the anthracycline anticancer drug doxorubicin and the nucleic acid dyes ethidium bromide (E) and 4',6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, a competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.

Quantitative detection method for Roundup Ready soybean in food using duplex real-time PCR MGB chemistry.

BACKGROUND: Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non-authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. RESULTS: Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. CONCLUSION: In this study, a duplex qRTPCR using TaqMan minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products.

Assessment of atypical DNA intercalating agents in biological and in silico systems.

Non-covalent genotoxic interaction between DNA and classical planar fused-ring intercalating agents, has been well understood for some time especially in the context of frameshift mutagenesis in bacterial systems. Recent evidence, however, suggests that a rather wide structural range of small non-fused ring molecules may also be capable of partial or complete DNA intercalation in mammalian cells. The present paper will review recent studies on the identification and characterization of such atypically-structured molecules utilizing both cell-based and three-dimensional computational analyses focusing principally on prediction and detection of these atypical molecules. Mechanistic aspects of genotoxicity of such non-covalent binding molecules, with emphasis on marketed pharmaceuticals, will also be discussed. A review and presentation of new data using catalytic DNA topo II inhibitors, confirms the notion that topoisomerase II poisoning arising via intercalation is the major mechanism of genotoxicity of these drugs.

Atomic force microscopy and drug discovery.

Atomic force microscopy is being used ever more widely in biological imaging, because of its unique ability to provide structural information at the single molecule level and under near-physiological conditions. Detailed topographic images of potential drug targets, such as proteins and DNA, have been produced, and the folding of modular proteins has been studied using single-molecule force spectroscopy. Recently, atomic force microscopy has been used to examine ligand-protein and ligand-DNA interactions, and to begin to determine the architecture of multi-subunit proteins, including a member of the superfamily of ionotropic receptors. Atomic force microscopy is fast becoming a valuable addition to the pharmaceutical industry's toolkit.

Synthesis of mono- and bisdihydrodipyridopyrazines and assessment of their DNA binding and cytotoxic properties.

Aminoalkyl-substituted monomeric and dimeric dihydrodipyridopyrazines have been synthesized and evaluated as antitumor agents. Potent cytotoxic compounds were identified in both series. Biochemical and biophysical studies indicated that all these compounds strongly stabilized the duplex structure of DNA and some of them elicited a selectivity for GC-rich sequences. Sequence recognition by of the dimeric dihydrodipyridopyrazines is reminiscent of that of certain antitumor bisnaphthalimides. Compared to monomers, corresponding dimeric derivatives showed higher affinity for DNA. This property was attributed to a bisintercalative binding to DNA. This assumption was indirectly probed by electric linear dichroism and DNA relaxation experiments. DNA provides a bioreceptor for these dihydrodipyridopyrazine derivatives, but no poisoning of human topoisomerases I or II was detected. Most of the compounds efficiently inhibited the growth of L1210 murine leukemia cells and perturbed the cell cycle progression (with a G2/M block in most cases). A weak but noticeable in vivo antitumor activity was observed with one of the dimeric compounds. This studies identifies monomeric and dimeric dihydrodipyridopyrazines as a new class of DNA-targeted antitumor agents.

High-resolution assessment of protein DNA binding affinity and selectivity utilizing a fluorescent intercalator displacement (FID) assay.

Protein titration displacement of ethidium bromide bound to hairpin deoxyoligonucleotides containing any sequence of interest provides a well-defined titration curve (measuring the loss of fluorescence derived from the DNA bound ethidium bromide) that provides both absolute binding constants (K(a)) and stoichiometry of binding. This use of a fluorescent intercalator displacement (FID) assay for establishing protein DNA binding affinity and selectivity is demonstrated with the examination of the LEF-1 HMG domain binding to hairpin deoxyoligonucleotides containing its commonly accepted consensus sequence 5'-CTTTGWW (W=A or T) and those modified (5'-CTNTGWW) to examine sequences implicated in early studies (5'-CTNTG). The effectiveness of the FID assay coupled with its technically non-demanding experimental use makes it an attractive alternative or complement to selection screening, footprinting or affinity cleavage, and electrophoretic mobility shift assays for detecting, characterizing, and quantitating protein DNA binding affinity and selectivity.

Assessment of the sequence dependency for the binding of 2-aminonaphthyridine to the guanine bulge.

We have studied the sequence dependent binding of 2-amino-1,8-naphthyridine derivative 1 to a single guanine bulge. The free energy changes for the binding to a guanine bulge with different sequence contexts (5'X_Y3'/3'X'GY'5') were determined by a curve fitting of the thermal denaturation profile of DNA in the presence and absence of 1. The data showed that (i) the binding of 1 to a guanine bulge is stronger for those flanking the G-C base pair than A-T base pair, (ii) the guanine 3' side to 1 in the complex is especially effective for the complex stabilization, and (iii) the increase of T(m) in the presence of 1 is not a good estimate for the sequence dependent binding. The most efficient 1-binding was observed for the sequence of G_G/CGC. Molecular modeling simulations suggested that stacking interaction between the 3' side guanine and 1 is the molecular basis for the strong binding to G_G/CGC.

Femtosecond electron-transfer reactions in mono- and polynucleotides and in DNA.

Quenching of redox active, intercalating dyes by guanine bases in DNA can occur on a femtosecond time scale both in DNA and in nucleotide complexes. Notwithstanding the ultrafast rate coefficients, we find that a classical, nonadiabatic Marcus model for electron transfer explains the experimental observations, which allows us to estimate the electronic coupling (330 cm(-1)) and reorganization (8070 cm(-1)) energies involved for thionine-[poly(dG-dC)](2) complexes. Making the simplifying assumption that other charged, pi-stacked DNA intercalators also have approximately these same values, the electron-transfer rate coefficients as a function of the driving force, DeltaG, are derived for similar molecules. The rate of electron transfer is found to be independent of the speed of molecular reorientation. Electron transfer to the thionine singlet excited state from DNA obtained from calf thymus, salmon testes, and the bacterium, micrococcus luteus (lysodeikticus) containing different fractions of G-C pairs, has also been studied. Using a Monte Carlo model for electron transfer in DNA and allowing for reaction of the dye with the nearest 10 bases in the chain, the distance dependence scaling parameter, beta, is found to be 0.8 +/- 0.1 A(-1). The model also predicts the redox potential for guanine dimers, and we find this to be close to the value for isolated guanine bases. Additionally, we find that the pyrimidine bases are barriers to efficient electron transfer within the superexchange limit, and we also infer from this model that the electrons do not cross between strands on the picosecond time scale; that is, the electronic coupling occurs predominantly through the pi-stack and is not increased substantially by the presence of hydrogen bonding within the duplex. We conclude that long-range electron transfer in DNA is not exceptionally fast as would be expected if DNA behaved as a "molecular wire" but nor is it as slow as is seen in proteins, which do not benefit from pi-stacking.

DNA cross-linking by azinomycin B: Monte Carlo simulations in the evaluation of sequence selectivity.

A new set of charges specifically developed for biologically relevant N7-alkylated purine adducts have been implemented in the AMBER force field of the MacroModel package and applied to the conformational search of azinomycin B-DNA interactions. To perform a sequence dependent reactivity relationship study, four DNA triplets known to interact differently with the drug, 5'-GCT-3', 5'-GCC-3', 5'-GTC-3', and 5'-GTT-3', have been modeled in B-form and intercalative conformations. Monte Carlo simulations of all possible monoadducts and intercalative complexes have been carried out and analyzed using a filtering criterion that estimates the probability of covalent bond formation and covalent cross-linking. We observed a good correlation between existing experimental data and our computational estimations that validate the approach. The comparison of the conformational properties of the drug-DNA monoadducts and complexes confirms the most probable mechanism of action involving an initial aziridine and subsequent epoxide alkylation. The different hydrogen bond network in the monoadducts and in the intercalative complexes between the drug and the three base-pair receptor is the primary reason for the different cross-linking reactivity. In addition, steric hindrance of the major groove exposed methyl group of central thymine-based triplets plays an important role in the lack of the reactivity of these sequences. Synthetic work on the azinomycins and the information coming from this computational study will be important for the design of more potent or DNA sequence-selective agents based on the azinomycin skeleton.

Structural differences between the free and bound states of the DNA-bisintercalating peptide YSPTSPSY.

The YSPTSPSY peptide is a DNA-bisintercalator that can adopt nonrandom conformations in solution. Strategies based on random conformational search and energy minimizations have been applied to generate populations of conformers characterizing YSPTSPSY. Subsequent analysis based on statistical methods and clustering allowed to determine the existence of four classes of conformers containing beta- and/or gamma-turns. NMR spectra of YSPTSPSY in solution provide evidence for such structures. Employing a Monte Carlo-based docking procedure, the YSPTSPSY peptide was docked in a DNA double-helical fragment with the sequence [d(GACGTC)]2. The peptide binds on the minor groove of DNA stacking the central CG base pairs, in a manner similar to that observed in complexes of triostin A with DNA. Upon binding, the structure of the C-terminal segment is modified into a type I beta-turn. Five intermolecular hydrogen bonds are observed, but the van der Waals interactions constitute the major stabilization factor for the complex. NMR chemical shifts, coupling constants, and NOESY connectivities are in agreement with the molecular model.

Assessment of fluorochromes for cellular structure and function studies by flow cytometry.

Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, such as fluorescence quenching and energy transfer, inherent to the staining, provide numerous applications in flow cytometry. The available fluorophores used in flow cytometry are classified according to their cellular incorporation and binding. Thus, probes are presented and discussed as follows: 1) dyes of cellular components (DNA, RNA, proteins, lipids); 2) probes of membrane potential; 3) fluorophores that are sensitive to their microenvironment (pH, calcium, etc); and 4) those used for measurement of enzymatic activities into cells.