Thermal Denaturation Studies

An abasic peptide (Ac-Gys -Lys-(Ser-Ala-Ala-Lys)4-Ser-Gly-Lys-NH2) with unmodified Ser residues was synthesized and used as a control. No induced cooperative binding to. TAjGsAsT) was observed in the UV melting curve with this abasic peptide. This indicated that the cooperative melting between aPNA and its complementary DNA was not merely a reflection of nonspecific interactions be- 

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Collectively, these thermal denaturation studies demonstrated that aPNAs bind to complementary ssDNA targets with high affinity and in a sequence-specific manner consistent with our proposed base-pairing model. Additional electrostatic and hydrophobic binding interactions can be incorporated into the aPNA design without affecting the primary Watson-Crick binding mode. 

Thermal denaturation studies on 15-mer oligomers indicated that 45 has a preference for base-pairing with dG compared to dA (Fig. 49)... 

Dialysis experiments and DNA thermal denaturation studies of bisimidazoletetrachloroirida-te(III), (250), suggest poor binding of (250) to DNA, with no formation of interstrand crosslinks.422... 

Finally, thermal denaturation studies of double stranded nucleic acids in the presence of the interacting species allow one also to draw conclusions on the intercalating abilities of a molecule [49] as the behaviour observed with classical intercalators and pure electrostatic binders are quite different. 

Hong, L., Schroth, G.P., Matthews, H.R., Yau, P., and Bradbury, E.M. (1993) Studies of the DNA binding properties of histone H4 amino terminus. Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 tail to DNA. J. Biol. Chem. 268, 305-314. 

Table I The melting temperatures (Tm) from the CD thermal denaturation studies are estimated from the temperature at which the slope of the first derivative of the uncorrected data was a minimum ty2 is the temperature of half-completion of the DSC thermal denaturation transition. A//cal is the calorimetric enthalpy. The DSC AAG°u = AG°u (mutant) - AG°u (wild-type), at the value of ti/2 for the wild-type protein (74.6°C).

One of the most important aspects of 4-fluoro-6-methylbenzimidazole as a modified nucleobase is its strong stabilization of duplex DNA through a stacking effect. The stacking properties of 97 and other modified nucleosides can be measured by the dangling end thermal denaturation studies of DNA duplexes with self-complementary stands (dXCGC-... 

The 5 -phosphoramidite of the novel bicyclic nucleoside (14), restricted to an S-type conformation, has been synthesised in eight steps via double inversion of configuration at the C4 position of protected l-(3 -deoxy-p-D-psicofuranosyl)uracyl and introduction of an azide moiety. The secondary amino group at C4 permitted the subsequent incorporation of (14) into oligonucleotides via a 5 -3 directed variation of the standard phosphoramidite approach for synthesis of oligonucleotides. Thermal denaturation studies showed rather large decreases in duplex stabilities toward complementary DNA and RNA. ... 

DSC studies of thermal denaturation studies of ribonuclease A (RNase A) showed a decrease in T values in ILs with various cations and anions as follows [11] ... 

Thermal denaturation studies of DNA and DNA-protein complexes are performed on a UV spectrophotometer equipped with temperature controlled cell holders. Melting of the DNA duplex is indicated by a cooperative hyperchromism at 260 nm. Stabilization by Sac7d is most readily observed with poly[d(AT)]-poly[d(AT)] because of its intrinsic low stability, especially in low salt. The UV melting curve of poly[d(AT)] poly[d(AT)] in 0.01 M K2HPO4 is sharp with a Tm of 43.5° (Fig. 9). In the presence of Sac7d, the melting profile of poly[d(AT)]-poly [d(AT)] broadens and the Tm increases by as much as 33°C for solutions with an excess of Sac7d. The observed Tm for the complex depends on the concentration of protein. 

In the 1950s, it became apparent that most RNA molecules, unlike DNA, are single-stranded (59). Hydrodynamic studies indicated that RNA molecules e usudly globular rather than extended like DNA. UV-monitored thermal denaturation studies showed that RNA molecules have considerable secondary structure as inferred from... 

The above data were obtained in solutions approximately 10 M, or a few micrograms per ml. When attempts were made to prepare solutions of higher concentrations,a heavy gummy precipitate was obtained. However, it one first forms a complex between poly-lysine and carboxymethylcellulose and then adds poly I poly C, a soluble material is obtained. This material, poly(ICLC), is thermodynamically more stable than poly I-poly C, as shown in thermal denaturation studies (Fig. 3). These studies were done in O.IX SSC, as poly(ICLC) did not "melt" below lOO C in SSC. The new compound is more resistant to hydrolysis by human sera or RNase than is poly I poly C (Fig. 4). Poly(ICLC) induces 5-10 times more interferon in mice than does poly I poly C (Levy et al., 1975a). 

Choice of buffer is an important yet often overlooked aspect of performing reliable UV thermal denaturation studies. Among the most important considerations are the buffer pK, buffer capacity, compatibility with divalent metals and temperature dependence of pKg. Table 1 lists the properties of the most commonly used buffers for UV melting experiments. A list of other non-complexing buffers over the entire pH range has recently been described (64). 

Table 1 Commonly used buffers for UV thermal denaturation studies ...

Tris Is not recommended for thermal denaturation studies due to the large temperature dependence of its PK,. 

Figure 6. Thermal denaturation studies of the MPEG-conjugated HIV-12mers first derivative of UV absorption at 260 nm as function of temperature.

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