DNA melting investigated by differential scanning calorimetry and Raman spectroscopy
DNA melting investigated by differential scanning calorimetry and Raman spectroscopy
复制标题
DOI：
10.1016/s0006-3495(96)79528-0
复制
发表时间：
1996-12-01
影响因子：
3.4
通讯作者：
Thomas, GJ
中科院分区：
文献类型：
--
作者：
Duguid, JG;Bloomfield, VA;Thomas, GJ
利用差示扫描量热法（DSC）和拉曼光谱对小牛胸腺DNA的160个碱基对（bp）片段进行了热变性研究。BSC结果表明，熔点的中位温度T-m = 75.5℃，热焓变δ H-vH = 6.7 kcal/mol (bp)，范特霍夫焓变δ H-vH = 50.4 kcal/mol（协同单位），热熵变δ S-cal = 19.3 cal/ g。mol (bp)，实验条件为55 mg DNA/ml，溶于pH 6.4的5 mM羧酸钠溶液中。平均协同熔化单位[n（熔体）]为7.5 bp。160bp DNA的拉曼特征对温度高度敏感。对几个构象敏感的拉曼光谱分析表明，熔解的热力学参数范围为43 < δ H-vH < 61 kcal/mol（协同单位），75 < T-m < 80℃，6 < [n（熔体）]< 9 bp，与DSC结果一致。特定拉曼带频率和强度随温度的变化表明，热变性伴随着沃森-克里克碱基对的破坏、碱基的解叠和B型主链的无序。在20 ~ 93℃的温度范围内，这三种类型的结构变化是高度相关的。拉曼光谱诊断了嘌呤和嘧啶的解叠，脱氧核糖-磷酸部分的构象重排以及磷酸基环境的变化。其中，834 cm(-1)（由于磷酸二酯基团的局部振动），1240 cm(-1)（胸腺嘧啶环）和1668 cm(-1) （dT， dG和dC的羰基）的波段与DSC结果比较显示为DNA熔化的最可靠的定量指标。相反，拉曼标记带在786 cm(-1)（胞嘧啶环）、1014 cm(-1)（脱氧核糖环）和1092 cm(-1)（磷酸基）的强度对熔化基本不变，并被建议作为强度归一化的适当标准。
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 BSC results indicate a median melting temperature T-m = 75.5 degrees C with calorimetric enthalpy change (Delta H-cal = 6.7 kcal/mol (bp), van't Hoff enthalpy change Delta H-vH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change Delta S-cal = 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 [n(melt)] 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 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 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.