Ratilainen T, Holmén A, Tuite E, Haaima G, Christensen L, Nielsen P E, Nordén B
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
Biochemistry. 1998 Sep 1;37(35):12331-42. doi: 10.1021/bi9808722.
The thermodynamics of hybridization and the conformations of decameric mixed purine-pyrimidine sequence PNA/PNA, PNA/DNA, and DNA/DNA duplexes have been studied using fluorescence energy transfer (FET), absorption hypochromicity (ABS), isothermal titration calorimetry (ITC), and circular dichroism (CD) techniques. The interchromophoric distances determined in the FET experiments on fluorescein- and rhodamine-labeled duplexes indicate that the solution structures of the duplexes are extended helices in agreement with available NMR (PNA/DNA) and crystal X-ray data (PNA/PNA). The melting thermodynamics of the duplexes was studied with both FET and ABS. The thermodynamic parameters obtained with ABS are in good agreement with the parameters from calorimetric measurements while FET detection of duplex melting gives in most cases more favorable free energies of hybridization. This discrepancy between FET and ABS detection is ascribed to the conjugated dyes which affect the stability of the duplexes substantially. Especially, the dianionic fluorescein attached via a flexible linker either to PNA or to DNA seems to be involved in an attractive interaction with the opposite dicationic lysine when hybridized to a PNA strand. This interaction leads to an increased thermal stability as manifested as a 3-4 degreesC increase of the melting temperature. For the PNA/DNA duplex where fluorescein is attached to the PNA strand, a large destabilization (DeltaTm = -12 degreesC) occurs relative to the unlabeled duplex, probably originating from electrostatic repulsion between the fluorescein and the negatively charged DNA backbone. In the case of the PNA/PNA duplex, the sense of helicity of the duplex is reversed upon conjugation of fluorescein via a flexible linker arm, but not when the fluorescein is attached without a linker to the PNA.
已使用荧光能量转移（FET）、吸收减色（ABS）、等温滴定量热法（ITC）和圆二色性（CD）技术研究了十聚体混合嘌呤 - 嘧啶序列PNA/PNA、PNA/DNA和DNA/DNA双链体的杂交热力学及构象。在对荧光素和罗丹明标记的双链体进行的FET实验中测定的发色团间距离表明，双链体的溶液结构是伸展的螺旋，这与现有的核磁共振（PNA/DNA）和晶体X射线数据（PNA/PNA）一致。用FET和ABS研究了双链体的解链热力学。用ABS获得的热力学参数与量热测量得到的参数高度一致，而FET检测双链体解链在大多数情况下给出更有利的杂交自由能。FET和ABS检测之间的这种差异归因于共轭染料，其对双链体的稳定性有显著影响。特别是，通过柔性接头连接到PNA或DNA上的二价阴离子荧光素在与PNA链杂交时似乎与相反的二价阳离子赖氨酸存在吸引相互作用。这种相互作用导致热稳定性增加，表现为解链温度升高3 - 4℃。对于荧光素连接到PNA链的PNA/DNA双链体，相对于未标记的双链体发生了很大的去稳定化（ΔTm = -12℃），这可能源于荧光素与带负电荷的DNA主链之间的静电排斥。在PNA/PNA双链体的情况下，通过柔性接头臂连接荧光素时双链体的螺旋方向会反转，但荧光素不通过接头连接到PNA时则不会。
