Thermal Stability Of DNA Duplexes Research Articles

THE STUDY OF THERMAL STABILITY OF DNA DUPLEX ON NANOPARTICLE-BASED PLATFORMS: A METHOD FOR BIOSENSING APPLICATIONS

In this research, a new method for DNA detection is proposed by studying the thermal stability of DNA duplexes modified on the nanostructured platform. The nanostructures are fabricated by using the block copolymer nanolithography method to form ordered arrays of gold nanoparticles on the silicon substrate. The thermally induced denaturation of the DNA duplex on the sensing part is monitored to determine the heat transfer property (thermal resistance) at the solid-liquid interface. An about 50% change in the thermal resistance during the DNA denaturation has fairly confirmed the feasible working of the sensing platform. The sensitivity, selectivity, and repeatability of the DNA biosensor are also evaluated by analysing the thermal resistance behaviour during thermal induction cycles. For the first time, a label-free, cost-effective, and easy data read-out DNA sensing method is developed in comparison with conventional fluorescence techniques.

PH-Dependence of the thermal stability of metallo-DNA duplexes containing ligand-type 5-hydroxyuracil nucleobases

Development of a metal-mediated base paring system has expanded the functions of DNA-based materials. We have recently reported that a 5-hydroyxuracil (UOH) nucleobase forms both a metal-mediated self-base pair (UOH–M–UOH; M=Gd(III) etc.) and a Watson–Crick-type hydrogen-bonded base pair with adenine nucleobase (UOH–A). In this study, we have investigated the pH-dependence of the thermal stability of DNA duplexes containing UOH–M–UOH base pairs or UOH–A base pairs in the absence or presence of Gd(III) or Zn(II) ions. Melting experiments and UV absorption analyses revealed that the complexation of UOH bases with Zn(II) ions altered the thermal stability of the DNA duplexes in a pH-dependent manner. In the presence of Zn(II) ions, a UOH–UOH-containing duplex was stabilized at higher pH while a UOH–A-containing duplex was destabilized. This pH-dependence of the metallo-DNA structures containing UOH nucleobases is specific to Zn(II) ions. Such dual responsiveness to both pH and metal ions would enrich switchable functions of metallo-DNA-based materials.

A DNA-based pattern classifier with in vitro learning and associative recall for genomic characterization and biosensing without explicit sequence knowledge.

BackgroundGenetic material extracted from in situ microbial communities has high promise as an indicator of biological system status. However, the challenge is to access genomic information from all organisms at the population or community scale to monitor the biosystem’s state. Hence, there is a need for a better diagnostic tool that provides a holistic view of a biosystem’s genomic status. Here, we introduce an in vitro methodology for genomic pattern classification of biological samples that taps large amounts of genetic information from all genes present and uses that information to detect changes in genomic patterns and classify them.ResultsWe developed a biosensing protocol, termed Biological Memory, that has in vitro computational capabilities to “learn” and “store” genomic sequence information directly from genomic samples without knowledge of their explicit sequences, and that discovers differences in vitro between previously unknown inputs and learned memory molecules. The Memory protocol was designed and optimized based upon (1) common in vitro recombinant DNA operations using 20-base random probes, including polymerization, nuclease digestion, and magnetic bead separation, to capture a snapshot of the genomic state of a biological sample as a DNA memory and (2) the thermal stability of DNA duplexes between new input and the memory to detect similarities and differences. For efficient read out, a microarray was used as an output method. When the microarray-based Memory protocol was implemented to test its capability and sensitivity using genomic DNA from two model bacterial strains, i.e., Escherichia coli K12 and Bacillus subtilis, results indicate that the Memory protocol can “learn” input DNA, “recall” similar DNA, differentiate between dissimilar DNA, and detect relatively small concentration differences in samples.ConclusionsThis study demonstrated not only the in vitro information processing capabilities of DNA, but also its promise as a genomic pattern classifier that could access information from all organisms in a biological system without explicit genomic information. The Memory protocol has high potential for many applications, including in situ biomonitoring of ecosystems, screening for diseases, biosensing of pathological features in water and food supplies, and non-biological information processing of memory devices, among many.Electronic supplementary materialThe online version of this article (doi:10.1186/1754-1611-8-25) contains supplementary material, which is available to authorized users.

A Comparative Study of the Influence of Aquaous 2,2,2-Trifluoroethanol And Ethanol on the Structural Organization, the Kinetic and Thermodynamic Properties of Oligodeoxyribonucleotides Intermolecular Complex Formation

A comparative study of the structural organization, thermodynamic and kinetic properties of the oligodeoxynucleotides complexes formation in the presence of 2,2,2-trifluoroethanol and ethanol in aqueous solution (volume fraction of alcohol 0 to 50 %) was performed. No significant changes in the circular dichroism spectra of oligonucleotides and their complexes at the adding of 50 % v/v alcohol into a solution, was observed, and they retain the profile typical for B-form DNA. The study of the thermal stability of DNA duplexes showed that the increase in the volume fraction of ethanol in the aqueous solution up to 50 % results in a linear decrease in the melting temperature of the intermolecular DNA complexes. In the case of the 2,2,2-trifluoroethanol we observed atypical dependence of thermal stability of DNA duplexes on the fraction of the fluorine-containing co-solvent. Increasing the alcohol fraction from 0 to 20% v/v led to a linear decrease of the melting point of the complex. A further increase in the volume fraction of alcohol (up to 50 %) did not change the thermal stability of the duplexes. It was shown, that the destabilizing effect of the two co-solvents is due to the increase of the dissociation rate constant of the complex and has mainly entropic nature. On the example of oligonucleotides complexes of 8, 12, 15 and 20 base pairs length the possibility of prediction DNA duplexes thermal stability was shown. A model taking into account the change of a number solvent molecules interacting with nucleic acids at the duplex formation in aqueous ethanol (50 % v/v) or trifluoroethanol (20 % v/v) was applied. An accuracy of melting temperature prediction was 1.3 and 0.6 degrees. Using this model, we found that the addition of alcohols in solution leads to an increase in the number of water molecules that bind to a complementary pair of nucleotides at the formation of intermolecular complex (in the presence of ethanol or trifluoroethanol 0.51 ± 0.09 and 1.33 ± 0.12, respectively). At the same time, alcohols interacted with single-stranded oligonucleotides and double-stranded in the same way

10.1007/s11171-008-2011-1

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.

Stacking interaction in the middle and at the end of a DNA helix studied with non-natural nucleotides

Base stacking is important for the base pair interaction of a DNA duplex, DNA replication by polymerases, and single-stranded nucleotide overhangs. To study the mechanisms responsible for DNA stacking interactions, we measured the thermal stability of DNA duplexes containing a non-natural nucleotide tethered to a simple aromatic hydrocarbon group devoid of dipole moments and hydrogen bonding sites. The duplexes containing tetrahydrofuran were paired with a deoxyadenosine derivative (A/T base pair analog) or a deoxycytidine derivative (C/G base pair analog) and showed a lower stability than Watson-Crick base pairing, partly due to the loss of interbase hydrogen bonds. Conversely, non-natural nucleotides present at a dangling end yielded an interaction energy as high as that observed with base pairing. Importantly, the non-natural nucleotides yielded an interaction energy with a linear correlation similar to that of the analogous Watson-Crick base pairs both in the middle and at the end of a DNA duplex, although a different stacking mechanism between the middle and the end was suggested. Moreover, a positive cooperativity was observed in dangling end stacking of the nucleotide base moiety and aromatic hydrocarbon group. These observations are useful to understand nucleic acid interactions and to design new non-natural nucleotides.

Effects of formamide on the thermal stability of DNA duplexes on biochips

In molecular biology, formamide (FA) is a commonly used denaturing agent for DNA. Although its influence on DNA duplex stability in solution is well established, little is known about immobilized DNA on microarrays. We measured thermal denaturation curves for oligonucleotides immobilized by two standard protocols: thiol self-assembling and pyrrole electrospotting. A decrease of the DNA denaturation temperature with increasing FA fraction of the solvent was observed on sequences with mutations for both surface chemistries. The average dissociation temperature decrease was found to be -0.58+/-0.05 degrees C/% FA (v/v) independently of grafting chemistry and probe sequence.

Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue into the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of the unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

Effective Modulation of DNA Duplex Stability by Reversible Transition Metal Complex Formation in the Minor Groove

Herein we describe the reversible changing of DNA duplex thermal stability by exploiting transition metal complexation phenomena. A terpyridine ligand was conjugated to the N2'-atoms of 2'-amino-2'-deoxyuridine and its locked counterpart 2'-amino-LNA, and these metal-complexing monomers were incorporated into oligodeoxyribonucleotides. Upon addition of varying amounts of transition metal ions, the thermal stability of DNA duplexes containing these terpyridine-functionalized units in different constitutions was affected to different degrees (DeltaTm values = -15.5 to +49.0 degrees C, relative to the unmodified duplex). The most pronounced effects were observed when two complexing monomers were positioned in opposite strands. Addition of 1 equiv of Ni2+ to such a system induced extraordinary duplex stabilization. Molecular modeling studies suggest, as an explanation for this phenomenon, formation of nickel-mediated interstrand linkages in the minor groove. Addition of an excess of metal ions resulted in largely decreased Tm values. Alternating addition of metal ions and EDTA demonstrated reversibility of metal ion-induced changes in hybridization strength, proving that the described approach provides an efficient method for duplex stability modulation.

Microfluidic Thermodynamics of the Shift in Thermal Stability of DNA Duplex in a Microchannel Laminar Flow

This paper reports the shift in thermal stability of DNA duplex and its thermodynamics spectroscopically, caused by stretching and orientation of DNA strands in a microchannel laminar flow. For direct spectroscopic measurement of the microchannel, we prepared an in-house temperature-controllable microchannel-type flow cell. The melting curves of DNA oligomers in a microchannel laminar flow were measured. For DNA oligomers with more than 10 base pairs, the melting curve shifted to the high-temperature side with higher flow speed. However, for 8-base-pair DNA oligomers, a change in the melting profile was not observed in batchwise and microchannel flows. We undertook microfluidic thermodynamic analysis to elucidate details of the shift in thermal stability of the DNA duplex in a microchannel laminar flow. Enthalpy-entropy compensation is applicable to the microfluidic thermal stability shift. We studied the relationships between the enthalpy-entropy compensation and DNA strand length or flow speed. Results showed that the enthalpy-entropy compensation was influenced by both DNA strand length and flow speed, and the penalties of enthalpy were 2-12% greater than the benefits of entropy.

A DNA Duplex with Extremely Enhanced Thermal Stability Based on Controlled Immobilization on Gold Nanoparticles

The effect of DNA loadings on the thermal stability of DNA duplex immobilized on gold nanoparticles has been investigated. The modestly loaded duplexes on the gold nanoparticles showed enhanced thermal stability, as compared to that of the free duplex (without gold nanoparticles). However, the highly loaded duplex showed stability similar to that of free duplex. The stability could be controlled over a wide temperature range simply by varying the salt concentration (over 50 degrees C). Additionally, the gold nanoparticles with modestly loaded oligonucleotides could be used as nanoprobes for effective and fast strand exchange reactions, based on the increased thermal stability of the immobilized duplex. These results indicate that the interaction between the duplex and the nanoparticle surface plays an important role in determining the stability of the duplex.

Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3′ terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes

Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3′ phosphodiesterase and 3′–5′ exonuclease activities. The 3′–5′ exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase β. Here, we examine the removal of matched and mismatched dNMP from the 3′ terminus of the 3′-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3′ terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3′ terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3′–5′ exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5′ margin of the nick. The 3′–5′ exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5′ margin of the nick as well as for flapped and recessed DNAs.

Effect of the Terminal Amino Group of a Linker Arm and Its Length at the C5 Position of a Pyrimidine Nucleoside on the Thermal Stability of DNA Duplexes

2′-Deoxyuridine derivatives bearing a substituent at the C5-position, which has a different chain length and a different functional group (methyl or amino), were synthesized and incorporated into oligodeoxyribonucleotides. The effect of the substituent groups in the major groove on the stability of the duplexes was investigated by UV melting experiments. It was found that the stabilization of these duplexes by a terminal amino group depended on the length of a linker arm.

Smoothing of the thermal stability of DNA duplexes by using modified nucleosides and chaotropic agents.

The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA duplexes has been investigated. We have shown that the use of tetramethylammonium ions TMA+along with the chemical modification of duplexes allow the fine adjustment of T m and the possibility of obtaining several duplex systems with varied isostabilizedtemperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization.

DNA DIVERGENCE AMONG HOMINOIDS.

We have determined the degree of single-copy DNA divergence among the extant members of the Hominoidea employing the technique of DNA-DNA hybridization. The species studied include humans, two species of chimpanzees, gorillas, two subspecies of orangutans, and two species of gibbons; as an outgroup we have used a member of the Old World monkeys (Cercopithecidae), the baboon. Our methods are different from those previously used and allow us to control for two factors other than base-pair mismatch that can affect the thermal stability of DNA duplexes: the base composition and duplex length. In addition, we have studied more than one individual for most species and thus are able to assess the effect of intraspecific variation on phylogenetic conclusions. The results indicate that the closest extant relatives of humans are the chimpanzees. Gorillas are the next closest, followed by orangutans and gibbons. This result is strongly supported statistically, as there is virtually no overlap in measurements between different taxa. Our conclusions are in agreement with a growing amount of molecular evidence supporting this pattern of relatedness. The data behave as a reasonably good molecular clock, and we do not see an indication of slowdown in molecular evolution in the clade containing humans and African apes, contrary to what has been documented for protein-coding regions. Because of the clocklike nature of the results, we have estimated that the divergence of humans and chimpanzees occurred about 6-8 million years ago. Results from orangutans indicate that the Borneo and Sumatra populations are genetically distinct, about as different as the named species of chimpanzees.

Calibration of the change in thermal stability of DNA duplexes and degree of base pair mismatch.

One method of determining the degree of base pair divergence between two sources of DNA (different strains, species, etc.) is to determine the decrease in thermal stability of hybrid duplex DNA due to mismatching of base pairs. Attempts to calibrate the change in median melting temperature (delta Tm) to base pair mismatch have led to conflicting results. We have studied the delta Tm between DNAs of known sequence over a range of from 0.55% to 7.2% base pair mismatch. The relationship of delta Tm and percent base pair mismatch is remarkably linear over this range with a correlation coefficient greater than 0.98. A delta Tm of 1 degree C corresponds to 1.7% base pair mismatch. This conversion is higher than that usually assumed and, therefore, rates of DNA evolution estimated by DNA-DNA hybridization studies are likely faster than previously thought.

Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.

Studies on nucleic acid interactions I. Stabilities of mini-duplexes (dG2A4XA4G2.dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases

The thermal stability of DNA duplexes containing deoxyinosine in a pairing position in turn with each of the four major deoxynucleotides has been investigated by measuring ultraviolet-absorbance at different temperatures. d(G2A4 X A4G2) and d(C2T4YT4C2) were prepared by the solid-phase phosphotriester method. When X is deoxyinosine, the Tm values of the duplexes are in the order Y = dC greater than dA greater than dG greater than dT greater than dU. The Tm of other duplexes containing dG, dA and dT at X were also measured. Self-complementary duplexes d(GGGAAINTTCCC) showed the same order of stability with N being dC, dA, dG and dT. Thermal stabilities of duplexes containing dG instead of dI were compared with other matched and mismatched duplexes. The Tm values of sequence isomers containing purine-pyrimidine combinations were compared. Self-complementary duplexes containing G-C and A-T in the central positions showed Tm values ca. 10 degrees higher than those containing C-G and T-A in the same positions. Thermodynamic parameters and circular dichroism spectra of these oligonucleotides were compared.

The relationship between mismatched base pairs and the thermal stability of DNA duplexes: II. Effects of deamination of cytosmine

The effect of mismatched base pairs on the thermal stability of renatured DNA · DNA duplexes was determined by specific alkali deamination of deoxycytosine residues to deoxyuridine residues. After annealing with unaltered DNA, the effect of the resulting G · U pairs on thermal stability was measured. The measurements of T m were made optically or by dissociation of filter- or hydroxylapatite-bound duplexes. The results suggest an effect such that 1 % G · U pairs reduces the thermal stability by about 2.2 °C. Considering the available data, an estimate is suggested for the effect of mismatching in heteroduplexes formed by naturally occurring DNA in which mutations have accumulated.

The relationship between mismatched base pairs and the thermal stability of DNA duplexes: I. Effects of depurination and chain scission

The effect of depurination and chain scission on the thermal stability of DNA · DNA duplexes was studied. Escherichia coli DNA was subjected to limited depurination followed by alkaline hydrolysis at the points of purine loss. These modified single-strands were annealed alone or in the presence of excess unmodified DNA. The thermal stability of the annealed duplexes was measured in three ways to permit estimation of the effect of the changes on the stability of the DNA double strands. The reduction in thermal stability is linearly related to the percentage of lesions and the slope of this relationship is approximately the same for depurination and for chain scission.