DNA Premelting Research Articles

Thermomechanical responses of microfluidic cantilever capture DNA melting and properties of DNA premelting states using picoliters of DNA solution

Melting or thermal denaturation of a DNA molecule and the different bubble-rich, premelting DNA states that serve as a precursor for DNA thermal denaturation are vital events in DNA thermophysics. In this study, we employ cantilever-based sensing to firstly pinpoint the occurrence of DNA melting and identify the temperature Tm characterizing the melting. Very importantly, this sensing is carried out with an extremely small volume (∼picoliters) of DNA sample with the cantilever demonstrating an extremely high sensitivity on the order of mJ/g⋅K corresponding to pico-Joules of energy input. Secondly, this same large sensitivity of the cantilever is used to quantify the hitherto unknown thermophysical properties of the bubble-rich DNA premelting states. In fact, for both the melting and premelting states, the cantilever provides a framework to calculate the specific heat capacity and the storage and loss moduli of the cantilever-DNA-solution system, thereby establishing a platform for quantifying DNAs' thermo-mechanical behavior.

Electrochemistry of Nucleic Acids

Publisher Summary The electroactivity of nucleic acids was discovered several years ago. It was shown that at mercury electrodes, adenine and cytosine were reduced in ssDNA while guanine produced an anodic signal due to the oxidation of guanine reduction product. The DNA signals at mercury electrodes are highly sensitive to changes in DNA structure due to DNA denaturation and renaturation, as well as to minor structural changes resulting from DNA premelting and DNA damage. The changes in DNA structure are reflected not only by the DNA faradaic responses but also by non-faradaic signals due to adsorption/desorption of DNA. Several interesting principles have been used in the development of the DNA sensors—such as amplified electrochemical analysis, investigation of charge transport, and tracing of changes in conformation of DNA. The s ensors for DNA hybridization and DNA damage lead the field of the electrochemistry of nucleic acids. The chapter briefly summarizes some properties of the elimination voltammetry with linear scan (EVLS) method. Oscillographic polarography, at controlled alternating current, was used in the electrochemical analysis of nucleic acids.

Temperature Control in Electrochemical DNA Sensing

This paper reviews reports that have considered temperature as an important parameter in electrochemical DNA detection. Only a couple of years after the electrochemical activity of DNA had been discovered in 1958, oscillopolarography in a thermostated cell was applied to study thermal behavior of DNA double strands. DNA premelting was discovered and denaturation curve analysis established. Later it was found that heated electrodes allow simple and easy control over temperature during accumulation, hybridization, dehybridization, and detection. Electrochemical melting curve analysis has been reported recently as an alternative to the classic UV-spectrophotometric counterpart. Most recent developments include electrochemical real time PCR. Keywords: DNA, electrochemical sensor, temperature, polymerase chain reaction, immobilization, hybridization, PCR amplification, thermocycler, UV-spectroscopy, spectroscopy

Temperature Control in Electrochemical DNA Sensing

This paper reviews reports that have considered temperature as an important parameter in electrochemical DNA detection. Only a couple of years after the electrochemical activity of DNA had been discovered in 1958, oscillopolarography in a thermostated cell was applied to study thermal behavior of DNA double strands. DNA premelting was discovered and denaturation curve analysis established. Later it was found that heated electrodes allow simple and easy control over temperature during accumulation, hybridization, dehybridization, and detection. Electrochemical melting curve analysis has been reported recently as an alternative to the classic UV-spectrophotometric counterpart. Most recent developments include electrochemical real time PCR. Keywords: DNA, electrochemical sensor, temperature, polymerase chain reaction, immobilization, hybridization, PCR amplification, thermocycler, UV-spectroscopy, spectroscopy

Physical studies of DNA premelting equilibria in duplexes with and without homo dA.cntdot.dT tracts: correlations with DNA bending

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.

Presence of nonlinear excitations in DNA structure and their relationship to DNA premelting and to drug intercalation.

We propose that collectively localized nonlinear excitations (solitons) exist in DNA structure. These arise as a consequence of an intrinsic nonlinear ribose inversion instability that results in a modulated beta alternation in sugar puckering along the polymer backbone. In their bound state, soliton-antisoliton pairs contain beta premelted core regions capable of undergoing breathing motions that facilitate drug intercalation. We call such bound state structures--beta premeltons. The stability of a beta premelton is expected to reflect the collective properties of extended DNA regions and to be sensitive to temperature, pH, ionic strength and other thermodynamic factors. Its tendency to localize at specific nucleotide base sequences may serve to initiate site-specific DNA premelting and melting. We suggest that beta premeltons provide nucleation centers important for RNA polymerase-promoter recognition. Such nucleation centers could also correspond to nuclease hypersensitive sites.

Structural and Energetic Considerations of Wave Propagation in DNA

An important dynamic aspect of DNA in solution is the presence of coupled motions in its structure that involve bending, stretching, unwinding, and shearing components. It is postulated that these motions arise from wave propagation in the polymer, excited through impulses generated by the random and continuous bombardment of DNA by solvent molecules along its length. This gives rise to travelling structural distortions in DNA that result in DNA breathing and in drug intercalation. These distortions can be thought of as premelting conformational changes in DNA, and, in this respect, could have important implications in understanding the structure of DNA active in RNA transcription, DNA replication, and genetic recombination. The nature of these distortions is further documented and their relationship to DNA breathing and to drug intercalation is discussed.A key intermediate in these processes is the multiply kinked structure, ..beta.. kinked DNA. This structure has both B and A characteristics, and it is postulated that this structure is a conformational intermediated in the B ..-->.. A polymorphic transition. In addition, a class of intercalators known as the bis functional intercalators, molecules that have two intercalative chromophores separated by about 10.2 A is discussed. These bifunctional intercalators appear to bind DNAmore » in a neighbor exclusion mode, and could, therefore, be probes to detect the migratory ..beta.. kinked DNA structure that has been postulated. The nature of the excitation force due to Brownian motion of solvent molecules that gives rise to wave motion in DNA is examined. This force is temperature dependent and one would therefore expect the average energy density along the DNA molecule to reflect this. As one raises the temperature of DNA in solution, additional localized regions of DNA premelting could arise. Events such as these could have important implications in understanding the mechanism of DNA melting.« less

Fluctuational opening of the double helix as revealed by theoretical and experimental study of DNA interaction with formaldehyde

It follows from the theory of helix-coil transition that local opening of the double helix due to thermal fluctuations must take place at temperatures well below the melting range. Provided that formaldehyde can not react with hydrogen-bonded bases and can react only with the exposed ones, such fluctuational opening of base-pairs may be probed by formaldehyde. To test the adequacy of the theory for describing the fluctuational opening of the double helix, the process of DNA interaction with formaldehyde has been simulated by the Monte Carlo method with the aid of a computer. On the basis of kinetic constants for the forward and reverse reactions of formaldehyde with all four nucleotides measured by McGhee & von Hippel (1975 a,b ), the kinetic curves of DNA unwinding by formaldehyde have been calculated theoretically without the use of any adjustable parameter. The calculations have demonstrated that the highly reversible but very fast reaction of formaldehyde with the imino group of thymine plays a very important role in the process of DNA unwinding by formaldehyde have been compared with experimental data obtained for bacteriophage T7 DNA for different values of pH, temperature and concentration of formaldehyde. This comparison leads to the conclusion that the theory offers a correct general description of fluctuational opening of the double helix. The main characteristics of this process calculated by the theory are as follows. At room temperatures only individual base-pairs are opened and the mean distance between adjacent unpaired bases is as great as 2×10 5 base-pairs. Each A·T pair is shown to be opened with frequency about 10 2 s −1 and each G·C pair with a frequency about 10 s −1 . At elevated temperatures, in the DNA premelting region, the probability of fluctuational opening, as well as the average number of base-pairs in an opened region, increases considerably. A possible role of the results obtained from an analysis of the processes of DNA function in the cell is discussed.