DNA melting investigated by differential scanning calorimetry and Raman spectroscopy

Abstract

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.

The Structure of Short and Genomic DNA at the Interparticle Junctions of Cationic Nanoparticles

“…We have previously shown that short DNA duplexes of 21 base pairs (dsDNA 21 ) mediate AgSp nanoparticle aggregation acting as electrostatic molecular linkers.…”

“…[16] As a result, dsDNA 21 behaves as a rigid rod of ≈7 nm length and 2 nm diameter, which is collected at the surface of individual quasispherical nanoparticles of around 22 nm diameter with minimal bending of the double helix. [10] Molecular dynamics simulations of dsDNA 21 adsorption on AgSp nanoparticles previously indicated that the duplex rapidly adheres onto the metal surface adopting an elongated conformation (for low surface coverage).…”

“…[11c] Consequently, interparticle gaps of ≈2.5 nm are generated in dsDNA 21 -mediated clusters (i.e., the diameter of the duplex rod-like structure sandwiched between spermine molecules bound to the metallic surfaces). It is also worth noting that, in addition to their physical size, DNA molecules coordinated at the metallic surfaces screen further adsorption of unbound duplexes by electrostatic repulsion.…”

Specific Applications of Vibrational Spectroscopy in Biomedical Engineering

“…Moreover, it is possible to monitor the denaturing processes (transition from ordered double stranded to a disordered single-stranded helix) and the B→A and B→Z conformational changes (Duguid et al, 1996;Erfurth & Peticolas, 1975;Prescott et al, 1984;Taillandier et al, 1989). Collected in Table 5 are the main Raman lines observed during the melting process and the B→A (Z) transitions.…”

“…Collected in Table 5 are the main Raman lines observed during the melting process and the B→A (Z) transitions. This technique also enables examination of the structural changes of DNA caused by radiation (Synytsya et al, 2007), temperature and pH change (Duguid et al, 1996;Erfurth & Peticolas, 1975;O'Connor et al, 1982) and the addition of metal ions or organic compounds (Duguid et al, 1993;Langlais et al, 1990;Martin et al, 1978;Martin et al, 1982). The Raman spectrum can be divided into characteristic spectral regions.…”