Melting Temperatures Of Duplexes Research Articles

Characterization of structural, genotoxic, and immunological effects of methyl methanesulfonate (MMS) induced DNA modifications: Implications for inflammation-driven carcinogenesis

Genotoxic DNA damaging agents are the choice of chemicals for studying DNA repair pathways and the associated genome instability. One such preferred laboratory chemical is methyl methanesulfonate (MMS). MMS, an SN2-type alkylating agent known for its ability to alkylate adenine and guanine bases, causes strand breakage. Exploring the outcomes of MMS interaction with DNA and the associated cytotoxicity will pave the way to decipher how the cell confronts methylation-associated stress. This study focuses on an in-depth understanding of the structural instability, induced antigenicity on the DNA molecule, cross-reactive anti-DNA antibodies, and cytotoxic potential of MMS in peripheral lymphocytes and cancer cell lines. The findings are decisive in identifying the hazardous nature of MMS to alter the intricacies of DNA and morphology of the cell. Structural alterations were assessed through UV–Vis, fluorescence, liquid chromatography, and mass spectroscopy (LCMS). The thermal instability of DNA was analyzed using duplex melting temperature profiles. Scanning and transmission electron microscopy revealed gross topographical and morphological changes. MMS-modified DNA exhibited increased antigenicity in animal subjects. MMS was quite toxic for the cancer cell lines (HCT116, A549, and HeLa). This research will offer insights into the potential role of MMS in inflammatory carcinogenesis and its progression.

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.

New Oligonucleotide 2'-O-Alkyl N3'→P5' (Thio)-Phosphoramidates as Potent Antisense Agents: Physicochemical Properties and Biological Activity.

We describe here the design, synthesis, physicochemical properties, and hepatitis B antiviral activity of new 2'-O-alkyl ribonucleotide N3'→P5' phosphoramidate (2'-O-alkyl-NPO) and (thio)-phosphoramidite (2'-O-alkyl-NPS) oligonucleotide analogs. Oligonucleotides with different 2'-O-alkyl modifications such as 2'-O-methyl, -O-ethyl, -O-allyl, and -O-methoxyethyl combined with 3'-amino sugar-phosphate backbone were synthesized and evaluated. These molecules form stable duplexes with complementary DNA and RNA strands. They show an increase in duplex melting temperatures of up to 2.5°C and 4°C per linkage, respectively, compared to unmodified DNA. The results agree with predominantly C3'-endo sugar pucker conformation. Moreover, 2'-O-alkyl phosphoramidites demonstrate higher hydrolytic stability at pH 5.5 than 2'-deoxy NPOs. In addition, the relative lipophilicity of the 2'-O-alkyl-NPO and NPS oligonucleotides is higher than that of their 3'-O- counterparts. The 2'-O-alkyl-NPS oligonucleotides were evaluated as antisense (ASO) compounds in vitro and in vivo using Hepatitis B virus as a model system. Subcutaneous delivery of GalNAc conjugated 2'-O-MOE-NPS gapmers demonstrated higher activity than the 3'-O-containing 2'-O-MOE counterpart. The properties of 2'-O-alkyl-NPS constructs make them attractive candidates as ASO suitable for further evaluation and development.

Metal-mediated DNA base pairing of easily prepared 2-oxo-imidazole-4-carboxylate nucleotides.

Metal-mediated DNA base pairs, which consist of two ligand-type artificial nucleobases and a bridging metal ion, have attracted increasing attention in recent years as a different base pairing mode from natural base pairing. Metal-mediated base pairing has been extensively studied, not only for metal-dependent thermal stabilisation of duplexes, but also for metal assembly by DNA templates and construction of functional DNAs that can be controlled by metals. Here, we report the metal-mediated base paring properties of a novel 2-oxo-imidazole-4-carboxylate (ImOC) nucleobase and a previously reported 2-oxo-imidazole-4-carboxamide (ImOA) nucleobase, both of which can be easily derived from a commercially available uridine analogue. The ImOC nucleobases were found to form stable ImOC–CuII–ImOC and ImOC–HgII–ImOC base pairs in the presence of the corresponding metal ions, leading to an increase in the duplex melting temperature by +20 °C and +11 °C, respectively. The ImOC bases did not react with other divalent metal ions and showed superior metal selectivity compared to similar nucleobase design reported so far. The ImOC–CuII–ImOC base pair was much more stable than mismatch pairs with other natural nucleobases, confirming the base pair specificity in the presence of CuII. Furthermore, we demonstrated the quantitative assembly of three CuII ions inside a DNA duplex with three consecutive ImOC–ImOC pairs, showing great potential of DNA-template based CuII nanoarray construction. The study of easily-prepared ImOC base pairs will provide a new design strategy for metal-responsive DNA materials.

Silver(I)‐mediated hetero base pairs of 6‐pyrazolylpurine and its deaza derivatives

AbstractA series of antiparallel‐stranded and parallel‐stranded DNA duplexes containing hetero base pairs of the artificial nucleobases 6‐(1H‐pyrazol‐1‐yl)‐9H‐purine (6PP), 1‐deaza‐6PP (1D6PP), 7‐deaza‐6PP (7D6PP), and 1,7‐dideaza‐6PP (1,7D6PP) were investigated with respect to their ability to form silver(I)‐mediated base pairs. A close examination of the duplex melting temperatures in the absence and in the presence of silver(I) ions shows a strong sequence‐dependence of the stability of the resulting silver(I)‐mediated base pairs. The most stable silver(I)‐mediated base pairs are formed when 1,7D6PP is located in a purine‐rich strand opposite either 6PP or 7D6PP. This indicates that the silver(I) ions are coordinated via the Watson‐Crick edge of the latter nucleobase, whereas they bind to the pyrazolyl moiety of the former. As a result, a [2+1] coordination environment is proposed for the silver(I) ions.

Light-induced formation of silver(I)-mediated base pairs in DNA: Possibilities and limitations

The applicability of the caged nucleoside CNPP (2′-deoxycytidine bearing an ortho-nitrophenylpropyl group at the exocyclic N4 position) in the light-triggered formation of three silver(I)-mediated base pairs was probed, namely C–Ag(I)–C, Im–Ag(I)–C and P–Ag(I)–C (Im = imidazole 2′-deoxyribonucleoside; P = nucleoside analogue with a 1H-imidazo[4,5-f][1,10]phenanthroline nucleobase surrogate). Generally, an increase in the duplex melting temperature was observed upon photo-deprotection in the presence of Ag(I), indicating the formation of the respective silver(I)-mediated base pair. Nevertheless, a complete photo-deprotection could not be achieved even under optimized conditions. A comparison of the results obtained for the three base pairs allows to derive some general conclusions regarding the optimal design of a metal-mediated base pair whose formation can be triggered by light.

Thermodynamics of DNA: heat capacity changes on duplex unfolding

The heat capacity change, ΔCp, accompanying the folding/unfolding of macromolecules reflects their changing state of hydration. Thermal denaturation of the DNA duplex is characterized by an increase in ΔCp but of much lower magnitude than observed for proteins. To understand this difference, the changes in solvent accessible surface area (ΔASA) have been determined for unfolding the B-form DNA duplex into disordered single strands. These showed that the polar component represents ~ 55% of the total increase in ASA, in contrast to globular proteins of similar molecular weight for which the polar component is only about 1/3rd of the total. As the exposure of polar surface results in a decrease of ΔCp, this explains the much reduced heat capacity increase observed for DNA and emphasizes the enhanced role of polar interactions in maintaining duplex structure. Appreciation of a non-zero ΔCp for DNA has important consequences for the calculation of duplex melting temperatures (Tm). A modified approach to Tm prediction is required and comparison is made of current methods with an alternative protocol.

Oligonucleotide⁻Palladacycle Conjugates as Splice-Correcting Agents.

2’-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5’-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human cervical cancer (HeLa Luc/705), human liver (HuH7_705), and human osteosarcoma (U-2 OS_705) reporter cell lines. Melting temperatures of duplexes formed by the modified oligonucleotides were approximately 5 °C lower than melting temperatures of the respective unmodified duplexes. The cyclopalladated oligonucleotides functioned as splice-correcting agents in the HeLa Luc/705 cell line somewhat more efficiently than their unmodified counterparts. Furthermore, the introduction of this chemical modification did not induce toxicity in cells. These results demonstrate the feasibility of using covalently metalated oligonucleotides as therapeutic agents.

Metal-mediated base pairing in DNA involving the artificial nucleobase imidazole-4-carboxylate

The use of imidazole-4-carboxylate (X) as an artificial nucleobase in metal-mediated base pairing is reported. Towards this end, the corresponding deoxyribonucleoside was synthesized and structurally characterized as its sodium salt (sodium 1,2-dideoxy-1-(4-carboxyimidazol-1-yl)-d-ribofuranose). The deoxyribonucleoside was incorporated into different DNA duplexes (parallel-stranded and antiparallel-stranded), and their Cu(II)- and Ag(I)-binding behavior was investigated. It was shown that both X–Cu(II)–X and X–Ag(I)–X base pairs can be formed, with the former being more stabilizing than the latter. The formation of an X–Cu(II)–X base pair is accompanied by an increase in the duplex melting temperature of approximately 20 °C for antiparallel-stranded duplexes and of 12 °C for the parallel-stranded duplex under investigation. Imidazole-4-carboxylate represents the first imidazole-based nucleoside for Cu(II)-mediated base pairing. Moreover, it is the smallest nucleoside known to form stable Cu(II)-mediated base pairs. Structures of the X–Cu(II)–X and X–Ag(I)–X base pairs are proposed, too, based on molecular structures obtained using the model nucleobase 1-benzyl-1H-imidazole-4-carboxylate.

Development of a simple coarse-grained DNA model for analysis of oligonucleotide complex formation

In this paper, we develop a coarse-grained nucleotide model for the purpose of simulating large-scale aptamer-based hydrogel network formation in future research. In the model, each nucleotide is represented by a single interaction site containing sugar, phosphate and base. Discontinuous molecular dynamics (DMD) simulations are performed to simulate formation and denaturation of oligonucleotide duplexes as a function of temperature. The simulated melting temperatures of oligonucleotide duplexes are calculated in simulations of systems with different sequences, lengths and concentrations of oligonucleotides, and compared to data from the OligoAnalyzer tool. The denaturation of oligonucleotide triplexes containing a hybridised structure of three different oligonucleotides is analysed using both simulations and experiments. The nucleotide model is found to be a good predictor of the oligonucleotide’s hybridised state for both duplexes and triplexes. This coarse-grained model has wide ranging applications in the development or optimisation of DNA-based technologies including DNA origami, DNA-enabled hydrogels and DNA-based biosensors.

Cationic comb-type copolymer promotes DNA assembly on gold nanoparticles while enhancing particle dispersibility

We previously reported that graft copolymers comprised of a cationic backbone and abundant grafts of hydrophilic dextran formed soluble interpolyelectrolyte complexes with anionic biopolymers and facilitated self-assembly and folding of the biopolymers, such as duplex formation of DNA and helical folding of a peptide. In this study, effects of the cationic graft copolymers on assembly of gold nanoparticles (AuNPs) and that of DNA on AuNPs were explored. While polylysine homopolymer caused aggregation of AuNP, the graft copolymer did not induce the aggregation as monitored by adsorption spectra. The highly-grafted copolymer at nano molar concentration was capable of suppressing AuNP aggregation induced by 3 M NaCl. Moreover, the copolymer did not cause aggregation of AuNPs whose surface were modified with oligonucleotides (ODN) having highly negative charges. In the presence of copolymer, melting temperature of DNA duplex formed between AuNP-ODN and its complementary ODN was increased about 10 °C, indicating that the copolymer enhanced stability of DNA duplex on the surface of AuNPs. It was concluded that the copolymer selectively promoted assembly of negatively charged DNA but inhibited aggregation of negatively charged AuNPs.

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection.

A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.

Metal-Dependent Nucleobase Recognition by Picolinamide.

A C-nucleoside featuring picolinamide as the base moiety has been synthesized and incorporated in the middle of a DNA oligonucleotide. The melting temperatures of duplexes formed by this modified oligonucleotide with its unmodified counterparts placing either adenine, cytosine, guanine or thymine opposite to the picolinamide residue were found to be highly dependent on the identity and concentration of transition metal ions in the samples. For example, AgI uniquely promoted pairing with adenine and PdII with guanine. Pairing with cytosine, on the other hand, was retarded by all of the metal ions studied (AgI , CuII , HgII , NiII , PdII and ZnII ).

Coarse-Grained Modeling of Nucleic Acids Using Anisotropic Gay–Berne and Electric Multipole Potentials

In this work, we attempt to apply a coarse-grained (CG) model, which is based on anisotropic Gay-Berne and electric multipole (EMP) potentials, to the modeling of nucleic acids. First, a comparison has been made between the CG and atomistic models (AMBER point-charge model) in the modeling of DNA and RNA hairpin structures. The CG results have demonstrated a good quality in maintaining the nucleic acid hairpin structures, in reproducing the dynamics of backbone atoms of nucleic acids, and in describing the hydrogen-bonding interactions between nucleic acid base pairs. Second, the CG and atomistic AMBER models yield comparable results in modeling double-stranded DNA and RNA molecules. It is encouraging that our CG model is capable of reproducing many elastic features of nucleic acid base pairs in terms of the distributions of the interbase pair step parameters (such as shift, slide, tilt, and twist) and the intrabase pair parameters (such as buckle, propeller, shear, and stretch). Finally, The GBEMP model has shown a promising ability to predict the melting temperatures of DNA duplexes with different lengths.

Effect of backbone chemistry on hybridization thermodynamics of oligonucleic acids: a coarse-grained molecular dynamics simulation study.

In this paper we study how varying oligonucleic acid backbone chemistry affects the hybridization/melting thermodynamics of oligonucleic acids. We first describe the coarse-grained (CG) model with tunable parameters that we developed to enable the study of both naturally occurring oligonucleic acids, such as DNA, and their chemically-modified analogues, such as peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). The DNA melting curves obtained using such a CG model and molecular dynamics simulations in an implicit solvent and with explicit ions match with the melting curves obtained using the empirical nearest-neighbor models. We use these CG simulations to then elucidate the effect of backbone flexibility, charge, and nucleobase spacing along the backbone on the melting curves, potential energy and conformational entropy change upon hybridization and base-pair hydrogen bond residence time. We find that increasing backbone flexibility decreases duplex thermal stability and melting temperature mainly due to increased conformational entropy loss upon hybridization. Removing charges from the backbone enhances duplex thermal stability due to the elimination of electrostatic repulsion and as a result a larger energetic gain upon hybridization. Lastly, increasing nucleobase spacing decreases duplex thermal stability due to decreasing stacking interactions that are important for duplex stability.

Immobilization-mediated reduction in melting temperatures of DNA–DNA and DNA–RNA hybrids: Immobilized DNA probe hybridization studied by SPR

Abstract The melting temperature of probe-target nucleic acid hybrids plays an important role in sensor performance. This study employed surface plasmon resonance (SPR) techniques to investigate the hybridization of both DNA and RNA targets to their complementary surface-bound DNA oligonucleotide and the corresponding melting temperatures. The target molecule was a 26 nucleotide strand of RNA selected because of its utility in detecting the tobacco mosaic virus, a common plant pathogen with an RNA genome. The melting temperatures of duplexes with immobilized probes were determined by hybridization of the DNA and RNA targets with the bound DNA probe at different temperatures using a custom-built thermostated dual-channel SPR cell. Hybridization conditions and binding efficiencies were compared for both DNA and RNA binding to the gold-coated surface-bound DNA probe. The melting temperature for the DNA–DNA duplex was approximately 15 °C higher than the DNA–RNA in the solid state. The melting temperature in solution was also measured for comparison to the surface values. For both RNA and DNA, the melting transitions were substantially lower (∼20 °C) for solid state binding relative to solution. Particularly for surface immobilized DNA–RNA hybrids, the melting temperature was reduced to room temperature or below. Since most biosensor platforms operate at room temperature, they may consequently exhibit poor sensor performance due to weakened probe-target interactions. This result emphasizes that for optimal sensor performance, the hybrid melting temperature should be considered. The ability to study and optimize the binding of complementary strands of genomic materials to bound DNA probes could facilitate the rapid detection and identification of plant viruses having genomic RNA using various biosensor rapid analytical techniques.

A Temperature-Controlled Microwell Platform for Electrochemical Melting Analyses

We have designed and fabricated a miniature device for proof-of-concept studies on biomolecular interactions using electrochemical melting analysis. A collection of temperature controlled three-electrode platforms was built on a 100-mm glass wafer, and it includes a total of eight separate probe sites. Each site consists of one 200 nm thick platinum (Pt) heater, one 200 nm gold (Au) electrode and two 200 nm Pt electrodes, as well as a 10 µL polydimethylsiloxane (PDMS) well, all used for probing electrochemical signals as a function of sample temperature. Our Pt heaters have the ability to control sample solution temperatures in the microwell from 4 °C to 120 °C. Heaters were fabricated with different geometries (width and shape) in order to select the best candidate for future device fabrication. Electrochemical melting analysis has been performed by detecting signals derived from changes in methylene blue (MB)-labeled DNA that occur during the heating process. The MB-labeled single strand(ss) DNA was added to hybridize with its matched thiolated ssDNA on Au electrode surface to form duplexes. Electron-transfer(eT) was monitored using square wave voltammetry (SWV) from the beginning of a thermal cycling process. During the heating process the eT was limited significantly when the heater temperature reached the duplex melting temperature. The dehybridized MB-labeled ssDNA left the Au electrode surface causing a current decrease. As a result, a current vs. temperature melting curve was obtained that allowed us to determine the sample DNA melting temperature. The rapid heating capability provided by the Pt heater allows the electrochemical melting measurements to be completed in 20-30 minutes, and that is significantly faster than the typical time for optical melting methods. Figure 1 illustrates the wired microsystem used in the DNA experiment, and includes an enlargement showing the configuration of the miniature electrodes and heater. We plan to build the described probes into a 3x3 array-based microchip for demonstrating the potential for high throughput DNA melting analyses to study biomolecular binding stability.

Structures, Dynamics, and Stabilities of Fully Modified Locked Nucleic Acid (β-d-LNA and α-l-LNA) Duplexes in Comparison to Pure DNA and RNA Duplexes

Locked nucleic acid (LNA) is a chemical modification which introduces a -O-CH2- linkage in the furanose sugar of nucleic acids and blocks its conformation in a particular state. Two types of modifications, namely, 2'-O,4'-C-methylene-β-D-ribofuranose (β-D-LNA) and 2'-O,4'-C-methylene-α-L-ribofuranose (α-L-LNA), have been shown to yield RNA and DNA duplex-like structures, respectively. LNA modifications lead to increased melting temperatures of DNA and RNA duplexes, and have been suggested as potential therapeutic agents in antisense therapy. In this study, molecular dynamics (MD) simulations were performed on fully modified LNA duplexes and pure DNA and RNA duplexes sharing a similar sequence to investigate their structure, stabilities, and solvation properties. Both LNA duplexes undergo unwinding of the helical structure compared to the pure DNA and RNA duplexes. Though the α-LNA substituent has been proposed to mimic deoxyribose sugar in its conformational properties, the fully modified duplex was found to exhibit unique structural and dynamic properties with respect to the other three nucleic acid structures. Free energy calculations accurately capture the enhanced stabilization of the LNA duplex structures compared to DNA and RNA molecules as observed in experiments. π-stacking interaction between bases from complementary strands is shown to be one of the contributors to enhanced stabilization upon LNA substitution. A combination of two factors, namely, nature of the -O-CH2- linkage in the LNAs vs their absence in the pure duplexes and similar conformations of the sugar rings in DNA and α-LNA vs the other two, is suggested to contribute to the stark differences among the four duplexes studied here in terms of their structural, dynamic, and energetic properties.

Facilitating Mismatch Discrimination by Surface-Affixed PNA Probes via Ionic Regulation

There has been a search for alternative nucleic acids that can be more effectively used in nucleic acid detection technologies compared to the DNA probes. Peptide nucleic acid (PNA), which contains a non-ionic peptidic backbone, offers such possibilities since it is nuclease-resistant, it binds to DNA with high affinity, and it can be readily self-assembled onto solid substrates, e.g., gold(111), with a molecular backbone orientation away from the substrate. Although application of PNA as a sensor probe has been exemplified, so far there is little or no account of the ionic modulation of single base mismatch discrimination capacity of surface-tethered PNA probes. Herein, we report "on-surface" melting temperatures of PNA-DNA duplexes formed on gold(111) surface, as obtained from fluorescence measurements. We show that surface-tethered PNA forms a stabler duplex than DNA, and is more effective in single base mismatch discrimination than DNA. Importantly, although PNA backbone is non-ionic, variation in the ionic components in hybridization buffer, i.e., varying concentration of monovalent sodium ion, and the nature of anion and the cation, exhibits clear effects on the mismatch discrimination capacity of PNA probes. In general, with decreasing cation concentration, PNA-DNA duplexes are stabilized and mismatch discrimination capacity of the PNA probes is enhanced. The stabilizing/destabilizing effects of anions are found to follow the Hofmeister series, emphasizing the importance of hydrophobic interaction between nucleobases for stability of the PNA-DNA duplexes. Interestingly, the nature of ionic dependence of "on-surface" mismatch detection ability of PNA probes differs significantly from the "solution" behavior of these probes.

Sequence-dependent thermodynamics of a coarse-grained DNA model

We introduce a sequence-dependent parametrization for a coarse-grained DNA model [T. E. Ouldridge, A. A. Louis, and J. P. K. Doye, J. Chem. Phys. 134, 085101 (2011)] originally designed to reproduce the properties of DNA molecules with average sequences. The new parametrization introduces sequence-dependent stacking and base-pairing interaction strengths chosen to reproduce the melting temperatures of short duplexes. By developing a histogram reweighting technique, we are able to fit our parameters to the melting temperatures of thousands of sequences. To demonstrate the flexibility of the model, we study the effects of sequence on: (a) the heterogeneous stacking transition of single strands, (b) the tendency of a duplex to fray at its melting point, (c) the effects of stacking strength in the loop on the melting temperature of hairpins, (d) the force-extension properties of single strands, and (e) the structure of a kissing-loop complex. Where possible, we compare our results with experimental data and find a good agreement. A simulation code called oxDNA, implementing our model, is available as a free software.