Duplex thermal stability

To summarize, the TEACL method allows for more accurate measurements of DNA duplex thermal stability while the HAP method, if automated, allows more replicates per unit effort. Finally, in the only case of which we are aware for which both the TEACL and HAP methods have been applied to the same taxa, the results are very similar [compare Sibley and Ahlquist (1987) with Caccone and Powell (1989)]. 

The thermal stability of the metal-containing DNA oligonucleotides proves that the incorporation of 9 into DNA oligonucleotides causes no destabilization of the duplex. 

Duplexes of two ENA strands or of one ENA strand and one DNA strand (RNA-DNA hybrids) can also be denatured. Notably, ENA duplexes are more stable than DNA duplexes. At neutral pH, denaturation of a doublehelical ENA often requires temperatures 20 °C or more higher than those required for denaturation of a DNA molecule with a comparable sequence. The stability of an RNA-DNA hybrid is generally intermediate between that of ENA and that of DNA The physical basis for these differences in thermal stability is not known. 

Data taken from N. Poklar, D. S. Pilch, S. J. Lippard, E. A. Redding, S. U. Dunham, and K. J. Breslauer, Influence of Cisplatin Intrastrand Crosslinking on the Conformation, Thermal Stability and Energetics of a 20-mer DNA Duplex, Proc. Natl. Acad. Sci. USA, 93, 7606-76U (1996). 

Fig. 5 Base pairing and base stacking free energy contributions to the helix thermal stability, as obtained from experiments with nicked duplexes, (a) Pairing shows negligible or even unfavorable effect on free energy, while stacking (b) gives favorable contribution with strong dependence on T, revealing both enthalpic and entropic effects. Adapted with permission from [9]...

Overall, the effects of polycations on the helix-coil transition of DNA are similar to those exhibited by the small cations both of them induce thermal stabilization of DNA duplex and decrease the cooperativity of the helix-coil transition In both cases the reasons for the stabilization effects are the screening of the negatively charged phosphate groups of the DNA, which reduces their electrostatic repulsion (Schildkraut, 1965). However, the effective concentration of a simple salt at which the same stabilization effect is achieved is ca. 103 times higher than the effective concentrations of a polycation. Consequently, polycations provide for a much more efficient screening of the DNA phosphate groups than the small cations. 

At high temperatures duplex dissociation occurs. The temperature at which 50% of the duplexes are dissociated is called the melting temperature Tm. The Tm value is, hence, a measure of the thermal stability of a duplex. How are TM values determined ... 

The thermal stability of a short DNA duplex can be estimated by the simple 4 + 2 rule each GC pair contributes 4° and each AT pair contributes 2° to the duplex melting temperature in 0.9 M NaCl solution.50 Although more rigorous estimates of duplex stability are possible,51 this simple method is reasonably accurate, and, in any event, we find it useful... 

Tm. The Melting Temperature (Tm) is a useful measure of thermal stability for small and intermediate divergences. The decrease in Tm with increasing divergence is steady until it reaches about the halfway point between criterion and the Tm of precise duplexes. At greater divergences, the fraction of DNA that hybridizes continues to drop steadily but the Tm decreases very little. This results in compression of ATm values at greater distances and is discussed elsewhere.7,9 35... 

An estimate of the extent of base-pair mismatch between hybridized DNA strands can be made by measuring the thermal stability of the reassociated duplexes. Place hybridized filters in fresh incubation buffer and heat at 5° increments to 90°. At each 5° increase, remove a small sample of the buffer and count. Controls are reassociated homologous DNAs. The amount of nucleotide mispairing has been estimated at 1% for each degree the Tm of the heterologous duplex is lowered (Arm).2 31... 

Recent reports indicate that divalent transition metal ions, such as Cu +, forming links between two artificial hydroxypyridone nucleobases can efficiently replace the hydrogen bonding between natural nucleobases, A-T and G-C, in oligonucleotides. Such artificial metal-mediated base pairs results in a moderate increase in the thermal stability of the duplex. They could lead to nucleic acid materials with novel chemical and physical properties. Such ohgonucleotide derivatives are of interest for the design of biosensors, nanomolecular wires, and switches. 

BNA (also known as LNA) has been used in TFOs in a pyrimidine motif at neutral pH. The binding constant of the BNA TFO was about 20 times larger than that of DNA as a result of a large decrease in the dissociation rate constant. A 3 -amino-2, 4 -BNA nucleotide has been prepared to introduce N3 -P5 phosphoramidate linkages. It is introduced as a dinucleotide unit (61), and in thermal stability studies was shown to exhibit superior duplex and triplex stability compared to either BNA or DNA, and shows enhanced resistance to digestion by SVPDE. These properties have previously been observed for N3 -P5 phosphoramidate linkages in DNA. ... 

A second structural requirement for the interferon-inducing capacity (antiviral activity) of synthetic polynucleotides is a stable, highly ordered secondary, hence double-stranded structure based on complementary base-pairing. The stability of the complex is reflected in its Tm (thermal stability) value. From a comparative study of different double-stranded RNA duplexes, De Clercq and Merigan61-1 and De Clercq et al. 53-) concluded that a Tm value higher than 60 °C (calculated for 0.15M Na+) was needed for full expression of antiviral activity. Since thermal stability represents a valuable measure of the overall stability of double-stranded RNAs116 double-stranded RNAs with Tm values higher than 60 °C may also be considered to be the most stable ones at 37 °C. 

Duplex Substitution1) of 2 -OH by Strand modified Molecular size (s20,w) Purine Pyrimidine strand strand Thermal stability (7m °C) 0.1 M Na+ 0.15 MNa+ Resistance of the modified strand to degradation by nucleases Interferon-inducing activity of the duplex References... 

Upon 98% acetylation of purine strand Tm °C was 44° upon 54% acetylation. 53° upon 30% acetylation, and 63° upon 0% acetylation of the purine strand83), Since substitution of 2 -0-C0-CH3 for 2 -OH in the poly(A) component of poly(A) poly(U) resulted in a significant decrease of the thermal stability of the duplex, the decrease in interferon-inducing activity brought about by the substitution should merely be regarded as a consequence of the decrease in thermal stability. 

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