Ratilainen T, Holmén A, Tuite E, Nielsen P E, Nordén B
Department of Physical Chemistry, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
Biochemistry. 2000 Jul 4;39(26):7781-91. doi: 10.1021/bi000039g.
For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (DeltaG degrees ) was determined to be -6.5+/-0.3 kJ mol(-1) bp(-1), corresponding to a microscopic binding constant of about 14 M(-1) bp(-1). A variety of single mismatches were introduced in 9- and 12-mer PNA-DNA duplexes. Melting temperatures (T(m)) of 9- and 12-mer PNA-DNA duplexes with a single mismatch dropped typically 15-20 degrees C relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only 0.02 M(-1) per mismatch. The impact of a mismatch was found to be dependent on the neighboring base pairs. To a first approximation, increasing the stability of the surrounding region, i.e., the distribution of A.T and G.C base pairs, decreases the effect of the introduced mismatch.
为了进一步表征肽核酸（PNA）的杂交特性，研究了混合序列PNA-DNA双链体杂交的热力学。我们主要使用吸收减色熔解曲线和等温滴定量热法，从结合亲和力（完全匹配的双链体）和结合的序列特异性（单个错配的双链体）方面对PNA与DNA的结合进行了表征。对于不同长度（6 - 20个碱基对）的完全序列匹配双链体，结合的平均自由能（ΔG°）被确定为 -6.5±0.3 kJ·mol⁻¹·bp⁻¹，对应于约14 M⁻¹·bp⁻¹的微观结合常数。在9聚体和12聚体PNA-DNA双链体中引入了多种单个错配。具有单个错配的9聚体和12聚体PNA-DNA双链体的熔解温度（Tm）相对于完全匹配序列通常下降15 - 20℃，相应的自由能损失约为15 kJ·mol⁻¹·bp⁻¹。因此，单个错配的平均代价估计约为两个匹配碱基对的增益或更大，导致每个错配的表观结合常数仅为0.02 M⁻¹。发现错配的影响取决于相邻碱基对。初步估计，增加周围区域的稳定性，即A·T和G·C碱基对的分布，会降低引入错配的影响。
