DNA MELTING INVESTIGATED BY DIFFERENTIAL SCANNING CALORIMETRY AND RAMAN-SPECTROSCOPY

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.