Jg. Duguid et al., DNA MELTING INVESTIGATED BY DIFFERENTIAL SCANNING CALORIMETRY AND RAMAN-SPECTROSCOPY, Biophysical journal, 71(6), 1996, pp. 3350-3360
Thermal denaturation of the B form of double-stranded DNA has been pro
bed by differential scanning calorimetry (DSC) and Raman spectroscopy
of 160 base pair (bp) fragments of calf thymus DNA. The BSC results in
dicate a median melting temperature T-m = 75.5 degrees C with calorime
tric enthalpy change (Delta H-cal = 6.7 kcal/mol (bp), van't Hoff enth
alpy change Delta H-vH = 50.4 kcal/mol (cooperative unit), and calorim
etric entropy change Delta S-cal = 19.3 cal/deg . mol (bp), at the exp
erimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6
.4. The average cooperative melting unit [n(melt)] comprises 7.5 bp. T
he Raman signature of 160 bp DNA is highly sensitive to temperature. A
nalyses of several conformation-sensitive Raman bands indicate the fol
lowing ranges for thermodynamic parameters of melting: 43 < Delta H-vH
< 61 kcal/mol (cooperative unit), 75 < T-m < 80 degrees C and 6 < [n(
melt)] < 9 bp, consistent with the DSC results. The changes observed i
n specific Raman band frequencies and intensities as a function of tem
perature reveal that thermal denaturation is accompanied by disruption
of Watson-Crick base pairs, unstacking of the bases and disordering o
f the B form backbone. These three types of structural change are high
ly correlated throughout the investigated temperature range of 20 to 9
3 degrees C. Raman bands diagnostic of purine and pyrimidine unstackin
g, 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) (ca
rbonyl groups of dT, dG and dC), are shown by comparison with DSC resu
lts to be the most reliable quantitative indicators of DNA melting. Co
nversely, the intensities of Raman marker bands at 786 cm(-1) (cytosin
e ring), 1014 cm(-1) (deoxyribose ring) and 1092 cm(-1) (phosphate gro
up) are largely invariant to melting and are proposed as appropriate s
tandards for intensity normalizations.