Nucleic acid melting

Crothers, D.M. (1971) Statistical thermodynamics of nucleic acid melting transitions with coupled binding equilibria. Biopolymers Vol. 10, pp. 2147-2160. 

C4H4N2O2. Colourless crystalline powder, turning brown at 280 C and melting at 338 C (decomp.). Uracil is a constituent of ribose nucleic acid. Used as a diuretic and derivatives have pharmaceutical importance. 5-Fluorouracil is used in cancer treatment. 

Myers, R.M., Fischer, S.G., Maniatis, T. and Lerman, L.S. (1985b) Modification of the melting properties of duplex DNA by attachment of a GC rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Research 13, 3111-3129. 

Delayed action cytotoxins that inhibit the synthesis of nucleic acids. They are obtained from various molds/fungi (Aspergillus flavus, Aspergillus parasiticus). They are colorless to pale-yellow crystalline materials melting above 450°F. The "B" toxins fluoresce blue in the presence of UV light while the "G" toxins fluoresce green. They are only slightly soluble in water, but are soluble in methanol, acetone, and chloroform. Aqueous solutions are "probably stable" and "probably tolerant" to chlorine at purification concentrations. 

Applications to fluorescent or fluorescently labeled proteins and nucleic acids, and to fluorescent lipid probes in phospholipid bilayers, have been reported. In the latter case, the diffusion coefficients measured above the chain melting temperature were found to be 10 7 cm2 s 1, which is in agreement with values obtained by other techniques. 

In 2004, Rayner and coworkers reported a dynamic system for stabilizing nucleic acid duplexes by covalently appending small molecules [34]. These experiments started with a system in which 2-amino-2 -deoxyuridine (U-NH ) was site-specifically incorporated into nucleic acid strands via chemical synthesis. In the first example, U-NH was incorporated at the 3 end of the self-complementary U(-NH2)GCGCA DNA. This reactive amine-functionalized uridine was then allowed to undergo imine formation with a series of aldehydes (Ra-Rc), and aldehyde appendages that stabilize the DNA preferentially formed in the dynamic system. Upon equilibration and analysis, it was found that the double-stranded DNA modified with nalidixic aldehyde Rc at both U-NH positions was amplified 34% at the expense of Ra and Rb (Fig. 3.16). The Rc-appended DNA stabilizing modification corresponded to a 33% increase in (melting temperature). Furthermore, imine reduction of the stabilized DNA complex with NaCNBH, resulted in a 57% increase in T. ... 

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

Melting Temperature. The double helix of polynucleotides described above becomes thermodynamically unstable at particular temperatures (with specified conditions of solute concentration, pH, etc.) and is transformed into the open random-coil arrangement. This transformation is rather sharp, and can be measured by the concurrent changes in a number of physical properties of the nucleic acid, such as the optical absorption coefficient. The midpoint of the transition region is called the melting point. 

Like proteins, nucleic acids can undergo denatur-ation. The strands of the double helix of DNA are separated and the double-stranded regions of RNA molecules "melt." Denaturation can be accomplished by addition of acids, bases, and alcohols or by removal of stabilizing counter ions such as Mg2+. The product is a random coil and denaturation can be described as a helix —> coil transition. Denaturation of nucleic acids by heat, like that of proteins, is cooperative (Chapter 7, Section A,3) and can be described by a characteristic melting temperature. 

A plot of the optical absorbance at 260 nm (the wavelength of maximum light absorption by nucleic acids) versus temperature is known as a melting curve (Fig. 5-45). The absorbance is lower, by up to 40%, for native than for denatured nucleic acids. This hypochromic effect (Chapter 23) is a result of the interaction between the closely stacked bases in the helices of the native molecules. The melting temperature Tm is taken as the midpoint of the increase in absorbance (Fig. 5-45). As the percentage of G + C increases, the nucleic acid becomes more stable toward denaturation because of the three hydrogen bonds in each GC pair. Tm increases almost linearly with increases in the G + C content. In the "standard" citrate buffer (0.15 M NaCl + 0.015 M sodium citrate, pH 7.0) Eq. 5-22 holds. The exact numerical relationship depends strongly upon the ionic composition and pH of the medium.37 72 552 553... 

Polymetaphosphate 3 Polymorphonuclear leukocytes 26 Polynucleotide(s) 199s, 226-228. See also Nucleic acids, RNA, DNA hybridization 255 hydrazine reactions 254 interactions with ions 218 melting of 255 probes 255... 

Whereas proteins have their low energy absorption band at 280 nm, polynucleotides typically have maxima at 260 nm (38,500 cm ). A phenomenon of particular importance in the study of nucleic acids is the hypochromic effect. In a denatured polynucleotide the absorption is approximately the sum of that of the individual components. However, when a double helical structure is formed and the bases are stacked together, there is as much as a 34% depression in the absorbance at 260 nm. This provides the basis for optical measurement of DNA melting curves (Fig. 5-45).45,86 The physical basis for the hypochromic effect is found in dipole-dipole interactions between the closely stacked base pairs.7,86,87... 

To determine whether the nucleic acid is single-stranded or double-stranded, it could be heated. A sharp increase in absorbance at 260 nm would indicate a double-stranded RNA or DNA a broader melting curve would suggest a single-stranded nu-... 

In addition to the base-pairing disruption, this melting also destroys the stacking of bases in fixed orientations relative to one another that is observed in the duplex. Later, we will present an argument based upon thermodynamic measurements of the stabilities of synthetic oligonucleotides (small pieces of synthetic nucleic acids) that the next-nearest neighbor interactions observed in the base stacking sequence are a major contributor to the relative stabilities of duplexes. 

Both the denaturation process in proteins and the melting transition (also referred to as the helix-to-coil transition) in nucleic acids have been modeled as a two-state transition, often referred to as the all-or-none or cooperative model. That is, the protein exists either in a completely folded or completely unfolded state, and the nucleic acid exists either as a fully ordered duplex or a fully dissociated monoplex. In both systems, the conformational flexibility, particularly in the high-temperature form, is great, so that numerous microstates associated with different conformers of the biopolymer are expected. However, the distinctions between the microstates are ignored and only the macrostates described earlier are considered. For small globular proteins and for some nucleic acid dissociation processes,11 the equilibrium between the two states can be represented as... 

Optical Methods and the van t Hoff Enthalpy The optical method relies upon the change in the UV absorption of both proteins and nucleic acids as the transformation from one molecular state to the other takes place. For example, the absorbance A at 260 nm of a solution containing a nucleic acid duplex is found to increase significantly from some baseline value as the duplex melts until it reaches a second baseline where the nucleic acid is completely melted. 

T. V. Chalikian, J. Volker, G. E. Plum, and K. J. Breslauer, A More Unified Picture for the Thermodynamics of Nucleic Acid Duplex Melting A Characterization by Calorimetric and Volumetric Techniques , Proc. Natl Acad. Sci. USA, 96, 7853-7858 (1999). 

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