VarGibbs Usage in the Optimization of Nearest-Neighbor Parameters and Prediction of Melting Temperature of RNA Duplexes.

The nearest-neighbor (NN) model is a general tool for the evaluation for oligonucleotide thermodynamic stability. It is primarily used for the prediction of melting temperatures but has also found use in RNA secondary structure prediction and theoretical models of hybridization kinetics. One of the key problems is to obtain the NN parameters from melting temperatures, and VarGibbs was designed to obtain those parameters directly from melting temperatures. Here we will describe the basic workflow from RNA melting temperatures to NN parameters with the use of VarGibbs. We start by a brief revision of the basic concepts of RNA hybridization and of the NN model and then show how to prepare the data files, run the parameter optimization, and interpret the results.

Temperature-Wise Calibration Increases the Accuracy of DNA Methylation Levels Determined by High-Resolution Melting (HRM).

High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT.

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

Machine learning based DNA melt curve profiling enables automated novel genotype detection.

Surveillance for genetic variation of microbial pathogens, both within and among species, plays an important role in informing research, diagnostic, prevention, and treatment activities for disease control. However, large-scale systematic screening for novel genotypes remains challenging in part due to technological limitations. Towards addressing this challenge, we present an advancement in universal microbial high resolution melting (HRM) analysis that is capable of accomplishing both known genotype identification and novel genotype detection. Specifically, this novel surveillance functionality is achieved through time-series modeling of sequence-defined HRM curves, which is uniquely enabled by the large-scale melt curve datasets generated using our high-throughput digital HRM platform. Taking the detection of bacterial genotypes as a model application, we demonstrate that our algorithms accomplish an overall classification accuracy over 99.7% and perform novelty detection with a sensitivity of 0.96, specificity of 0.96 and Youden index of 0.92. Since HRM-based DNA profiling is an inexpensive and rapid technique, our results add support for the feasibility of its use in surveillance applications.

Atomic force microscopy investigation of DNA denaturation on a highly oriented pyrolytic graphite surface.

Understanding of DNA interaction with carbonaceous surfaces (including graphite, graphene and carbon nanotubes) is important for the development of DNA-based biosensors and other biotechnological devices. Though many issues related to DNA adsorption on graphitic surfaces have been studied, some important aspects of DNA interaction with graphite remain unclear. In this work, we use atomic force microscopy (AFM) equipped with super-sharp cantilevers to analyze the morphology and conformation of relatively long DNA molecule adsorbed on a highly oriented pyrolytic graphite (HOPG) surface. We have revealed the effect of DNA embedding into an organic monolayer of N,N'-(decane-1,10-diyl)-bis(tetraglycinamide) (GM), which may "freeze" DNA conformation on a HOPG surface during drying. The dependence of the mean squared point-to-point distance on the contour length suggests that DNA adsorbs on a bare HOPG by a "kinetic trapping" mechanism. For the first time, we have estimated the unfolded fraction of DNA upon contact with a HOPG surface (24 ± 5 %). The obtained results represent a novel experimental model for investigation of the conformation and morphology of DNA adsorbed on graphitic surfaces and provide with a new insight into DNA interaction with graphite.

Cold denaturation of DNA origami nanostructures.

The coupling of structural transitions to heat capacity changes leads to destabilization of macromolecules at both elevated and lowered temperatures. DNA origami not only exhibit this property but also provide a nanoscopic observable of cold denaturation processes by directing intramolecular strain to the most sensitive elements within their hierarchical architecture.

Force-induced unzipping of DNA in the presence of solvent molecules.

The melting of double-stranded DNA (dsDNA) in the presence of solvent molecules is a fundamental process with significant implications for understanding the thermal and mechanical behavior of DNA and its interactions with the surrounding environment. The solvents play an essential role in the structural transformation of DNA subjected to a pulling force. In this study, we simulate the thermal and force induced denaturation of dsDNA and elucidate the solvent dependent melting behavior, identifying key factors that influence the stability of DNA melting in presence of solvent molecules. Using a statistical model, we first find the melting profile of short heterogeneous DNA molecules in the presence of solvent molecules in Force ensemble. We also investigate the effect of solvent's strengths on the melting profile of DNA. In the force ensemble, we consider two homogeneous DNA chains and apply the force on different locations along the chain in the presence of solvent molecules. Different pathways manifest the melting of the molecule in both ensembles, and we found several interesting features of melting DNA in a constant force ensemble, such as lower critical force when the chain is pulled from the base pair close to a solvent molecule. The results provide new insights into the force-induced unzipping of DNA and could be used to develop new methods for controlling the unzipping process. By providing a better understanding of melting and unzipping of dsDNA in the presence of solvent molecules, this study provides valuable guidelines for predicting DNA thermodynamic quantities and for designing DNA nanostructures.

Atomistic simulations of RNA duplex thermal denaturation: Sequence- and forcefield-dependence.

Double-stranded RNA is the end-product of template-based replication, and is also the functional state of some biological RNAs. Similarly to proteins and DNA, they can be denatured by temperature, with important physiological and technological implications. Here, we use an in silico strategy to probe the thermal denaturation of RNA duplexes. Following previous results that were obtained on a few different duplexes, and which nuanced the canonical 2-state picture of nucleic acid denaturation, we here specifically address three different aspects that greatly improve our description of the temperature-induced dsRNA separation. First, we investigate the effect of the spatial distribution of weak and strong base-pairs among the duplex sequence. We show that the deviations from the two-state dehybridization mechanism are more pronounced when a strong core is flanked with weak extremities, while duplexes with a weak core but strong extremities exhibit a two-state behavior, which can be explained by the key role played by base fraying. This was later verified by generating artificial hairpin or circular states containing one or two locked duplex extremities, which results in an important reinforcement of the entire HB structure of the duplex and higher melting temperatures. Finally, we demonstrate that our results are little sensitive to the employed combination of RNA and water forcefields. The trends in thermal stability among the different sequences as well as the observed unfolding mechanisms (and the deviations from a two-state scenario) remain the same regardless of the employed atomistic models. However, our study points to possible limitations of recent reparametrizations of the Amber RNA forcefield, which sometimes results in duplexes that readily denature under ambient conditions, in contradiction with available experimental results.

Properties of phosphoramide benzoazole oligonucleotides (PABAOs). I. Structure and hybridization efficiency of N-benzimidazole derivatives.

In this work, we for the first time conducted a detailed study on the structure, dynamics, and hybridization properties of N-benzimidazole group-bearing phosphoramide benzoazole oligonucleotides (PABAOs) that we developed recently. By circular dichroism we established that the introduction of the modifications does not disrupt the B conformation of the DNA double helix. The formation of complexes is approximated by a two-state model. Complexes of PABAOs with native oligodeoxriboynucleotides form efficiently, and the introduction of such modifications reduces thermal stability of short duplexes (8-10 bp) by ∼5°Ð¡ per modification. Using UV-spectroscopy analysis, a neutral charge of the phosphate residue modified by the N-benzimidazole moiety in the pH range of 3-9.5 was found. The results confirm possible usefulness of PABAOs for both basic research and biomedical applications.

Equilibrium melting probabilities of a DNA molecule with a defect: An exact solution of the Poland-Scheraga model.

In this study we derive analytically the equilibrium melting probabilities for basepairs of a DNA molecule with a defect site. We assume that the defect is characterized by a change in the Watson-Crick basepair energy of the defect basepair, and in the associated two stacking energies for the defect, as compared to the remaining parts of the DNA. The defect site could, for instance, occur due to DNA basepair mismatching, cross-linking, or by the chemical modifications when attaching fluorescent labels, such as fluorescent-quencher pairs, to DNA. Our exact solution of the Poland-Scheraga model for DNA melting provides the probability that the labeled basepair, and its neighbors, are open at different temperatures. Our work is of direct importance, for instance, for studies where fluorophore-quencher pairs are used for studying single basepair fluctuations of designed DNA molecules.

The binding of a c-MYC promoter G-quadruplex to neurotransmitters: An analysis of G-quadruplex stabilization using DNA melting, fluorescence spectroscopy, surface-enhanced Raman scattering and molecular docking.

The interactions of several neurotransmitter and neural hormone molecules with the c-MYC G-quadruplex DNA sequence were analyzed using a combination of spectroscopic and computational techniques. The interactions between indole, catecholamine, and amino acid neurotransmitters and DNA sequences could potentially add to the understanding of the role of G-quadruplex structures play in various diseases. Also, the interaction of the DNA sequence derived from the nuclear hypersensitivity element (NHE) III1 region of c-MYC oncogene (Pu22), 5'-TGAGGGTGGGTAGGGTGGGTAA-3', has added significance in that these molecules may promote or inhibit the formation of G-quadruplex DNA which could lead to the development of promising drugs for anticancer therapy. The results showed that these molecules did not disrupt G-quadruplex formation even in the absence of quadruplex-stabilizing cations. There was also evidence of concentration-dependent binding and high binding affinities based on the Stern-Volmer model, and thermodynamically favorable interactions in the form of hydrogen-bonding and interactions involving the π system of the aromatic neurotransmitters.

Aberrant methylation scanning by quantitative DNA melting analysis with hybridization probes as exemplified by liquid biopsy of SEPT9 and HIST1H4F in colorectal cancer.

OBJECTIVE: The generally accepted method of quantifying hypermethylated DNA by qPCR using methylation-specific primers has the risk of underestimating DNA methylation and requires data normalization. This makes the analysis complicated and less reliable. METHODS: The end-point PCR method, called qDMA-HP (for quantitative DNA Melting Analysis with hybridization probes), which excludes the normalization procedure, is multiplexed and quantitative, has been proposed. qDMA-HP is characterized by the following features: (i) asymmetric PCR with methylation-independent primers; (ii) fluorescent dual-labeled, self-quenched probes (commonly known as TaqMan probes) covering several interrogated CpGs; (iii) post-PCR melting analysis of amplicon/probe hybrids; (iv) quantitation of unmethylated and methylated DNA alleles by measuring the areas under the corresponding melt peaks. RESULTS: qDMA-HP was tested in liquid biopsy of colorectal cancer by evaluating SEPT9 and HIST1H4F methylations simultaneously in the single-tube reaction. Differences in the methylation levels in healthy donors versus cancer patients were statistically significant (p < 0.0001), AUCROC values were 0.795-0.921 for various marker combinations. CONCLUSIONS: This proof-of-concept study shows that qDMA-HP is a simple, normalization-independent, quantitative, multiplex and "closed tube" method easily adapted to clinical settings. It is demonstrated, for the first time, that HIST1H4F is a perspective marker for liquid biopsy of colorectal cancer.

Thermodynamic Characterization of Nucleic Acid Nanoparticles Hybridization by UV Melting.

The advances in nucleic acid nanotechnology have given rise to various elegantly designed structural complexes fabricated from DNA, RNA, chemically modified RNA strands, and their mixtures. The structural properties of NA nanoparticles (NANP) generally dictate and significantly impact biological function; and thus, it is critical to extract information regarding relative stabilities of the different structural forms. The adequate stability assessment requires knowledge of thermodynamic parameters that can be empirically derived using conventional UV-melting technique. The focus of this chapter is to describe methodology to evaluate thermodynamic data of NANPs complexation based on DNA 12 base-pair (bp) duplex formation as an example.

Force induced DNA melting in the presence of an attractive surface.

The self avoiding walk (SAW) model of the polymer has been extended to study the equilibrium properties of double stranded DNA (dsDNA) where two strands of the dsDNA are modeled by two mutually attracting self-avoiding walks (MASAWs) in the presence of an attractive surface. We study simultaneous adsorption and force induced melting transitions and explore different phases of DNA. It is observed that melting is entropically dominated, which can be substantially reduced under the application of an applied force. We consider three scenarios, where the surface is weakly, moderately and highly attractive. For both weakly and moderately attractive surfaces, the DNA desorbs from the surface in a zipped form and acquires the conformation of a melted state with the rise in temperature. However, for a strongly attractive surface, the force applied at one end of the strand (strand-II) results in unzipping, while the other strand (strand-I) remains adsorbed on the surface. We identify this as adsorption-induced unzipping, where the force applied on a single strand (strand-II) can unzip the dsDNA if the surface interaction energy exceeds a specific threshold. We also note that at a moderate surface attraction, the desorbed-zipped DNA melts with an increase in temperature and the free strand (strand-I) gets re-adsorbed onto the surface.

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity.

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

C-Banding of Plant Chromosomes.

C-banding visualizes regions of chromosomes containing constitutive heterochromatin. It creates distinct patterns along the chromosome length and allows precise chromosome identification if C-bands are present in sufficient numbers. It is performed on chromosome spreads generated from fixed material, usually root tips or anthers. While there are numerous lab-specific modifications, all methods share the same steps: acidic hydrolysis, DNA denaturation in strong bases (usually saturated aqueous solution of barium hydroxide), washes in saline solution, and staining in Giemsa-type stain in a phosphate buffer. The method can be used for a wide range of cytogenetic tasks, from karyotyping, meiotic chromosome pairing analyses, to large-scale screening and selection of specific chromosome constructs.

Toward a Molecular Mechanism of Complementary RNA Duplexes Denaturation.

RNA duplexes are relatively rare but play very important biological roles. As an end-product of template-based RNA replication, they also have key implications for hypothetical primitive forms of life. Unless they are specifically separated by enzymes, these duplexes denature upon a temperature increase. However, mechanistic and kinetic aspects of RNA (and DNA) duplex thermal denaturation remain unclear at the microscopic level. We propose an in silico strategy that probes the thermal denaturation of RNA duplexes and allows for an extensive conformational space exploration along a wide temperature range with atomistic precision. We show that this approach first accounts for the strong sequence and length dependence of the duplexes melting temperature, reproducing the trends seen in the experiments and predicted by nearest-neighbor models. The simulations are then instrumental at providing a molecular picture of the temperature-induced strand separation. The textbook canonical "all-or-nothing" two-state model, very much inspired by the protein folding mechanism, can be nuanced. We demonstrate that a temperature increase leads to significantly distorted but stable structures with extensive base-fraying at the extremities, and that the fully formed duplexes typically do not form around melting. The duplex separation therefore appears as much more gradual than commonly thought.

In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses.

Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. This research aimed to explore the ability to predict HRM outputs coupled to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To approach this goal a pair of degenerate genus-specific primers were designed for endpoint RT-PCR and RT-qPCR-HRM and the species in the genus Emaravirus were selected to framework the development of the assays. Both nucleic acid amplification methods were able to detect in-vitro several members of seven Emaravirus species with sensitivity up to one fg of cDNA. Specific parameters for in-silico prediction of the melting temperatures of each expected emaravirus amplicon are compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The high-resolution DNA melting curves of the RT-PCR products predicted in-silico using uMeltSM allowed saving time while designing and developing the RT-qPCR-HRM assay since the approach avoided extensive searching for optimal HRM assay regions and rounds of HRM tests in-vitro for optimization. The resultant assay provides sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains.

Pulling short DNA with mismatch base pairs.

Due to misincorporation during gene replication, the accuracy of the gene expression is often compromised. This results in a mismatch or defective pair in the DNA molecule (James et al. 2016). Here, we present our study of the stability of DNA with defects in the thermal and force ensembles. We consider DNA with a different number of defects from 2to16 and study how the denaturation process differs in both ensembles. Using a statistical model, we calculate the melting point of the DNA chain in both the ensemble. Our findings display different manifestations of DNA denaturation in thermal and force ensembles. While the DNA with defects denatures at a lower temperature than the intact DNA, the point from which the DNA is pulled is important in force ensemble.

Choline ester based ionic liquid: A multi-functional system to enhance nucleic acid stability, drug solubilization and cell penetration.

Ionic liquids (ILs) are emerging systems with applications in varying areas of biomedical research. This study aims at developing a biocompatible, dual function choline ester-based IL with chloride as anion ([Ch] IL) for stabilizing nucleic acids (DNA) and enhancing cellular uptake of drugs. The ability of IL to complex with DNA was characterized using electrophoresis, dye displacement and UV absorbance. The effect of pH on complex stability and protection of DNA from nuclease were also studied. Even though [Ch] IL had positive zeta potential and showed effective complex formation, at physiological pH the zeta potential of the complex decreased and became negative, thereby, destabilizing the complex. To address this, citric acid (CA) was added to [Ch] IL which facilitated strong complexation. Further, DNA could be retrieved from these complexes without compromising its purity and integrity. Additionally, [Ch] IL was found to improve the cellular uptake of doxorubicin by improving its solubility in water. Thus, we demonstrate that the [Ch] IL developed here can enhance nucleic acid stability, drug solubilization and cell penetration. Our results show that the developed [Ch] IL can be used for long term storage of nucleic acids as well as for enhancing permeation of drugs in vivo.