Abstract：
Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2═O stretching vibration when it is within the DNA duplex via isotopic substitutions at other atomic positions in the structure. First, we determined that this stretching state was localized enough to specific atoms in the molecule to make submolecular scale measurements of local structure and stability in high molecular weight complexes. Next, we develop a new isotope-edited variable temperature infrared method to measure melting transitions at various locations in a DNA structure. As an initial test of this \"sub-molecular scale thermometer\", we applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair near the end of an A-T rich sequence (poly T) is ∼34.9 ± 0.7°C. This is slightly lower than the TmL of a single base pair near the middle position of the poly T sequence (TmL ∼35.6±0.2°C). In addition, we also report that the TmL of a single Watson-Crick A-T base pair near the end of a 50% G-C sequence (12-mer) is ∼52.5 ± 0.3°C, which is slightly lower than the global melting Tm of the 12-mer sequence (TmL ∼54.0±0.9°C). Our results provide direct physical evidence for end fraying in DNA sequences with our novel spectroscopic methods.
摘要：
尽管对这些热力学参数非常感兴趣，但仍缺乏确定DNA双链体中单个碱基对解链事件的转变温度的实验方法。这里,我们通过结构中其他原子位置的同位素取代确定胸腺嘧啶(T)C2=O拉伸振动在DNA双链体中时的尺寸。首先,我们确定，这种拉伸状态是局部化的，足以在分子中的特定原子，使亚分子尺度测量的局部结构和稳定性在高分子量的配合物。接下来,我们开发了一种新的同位素编辑可变温度红外方法来测量DNA结构中各个位置的熔解转变。作为这个“亚分子尺度温度计”的初步测试，我们应用T13C2差异红外信号,通过可变温度衰减全反射傅里叶变换红外(VT-ATR-FTIR)光谱法测量DNA双链体的位置相关解链温度(TmL).我们报告，靠近富含A-T序列（polyT）末端的单个Watson-CrickA-T碱基对的TmL为〜34.9±0.7°C。这略低于polyT序列中间位置附近的单个碱基对的TmL（TmL〜35.6±0.2°C）。此外,我们还报告，接近50%G-C序列（12-mer）末端的单个Watson-CrickA-T碱基对的TmL为~52.5±0.3°C，略低于12聚体序列的全局解链Tm(TmL~54.0±0.9°C)。我们的结果为我们新颖的光谱方法在DNA序列中的末端磨损提供了直接的物理证据。
