Max-Planck-Institute for Polymer Research, D-55128 Mainz, Germany.
Biointerphases. 2007 Jun;2(2):80-8. doi: 10.1116/1.2746871.
Peptide nucleic acids (PNAs) are mimics of oligonucleotides containing a neutral peptidelike backbone and are able to bind complementary DNA targets with high affinity and selectivity. In order to investigate the effect of the ionic strength of the buffer solution, hybridization experiments with PNAs as (catcher) probes and DNAs as target oligonucleotides were performed in different salt solutions. Surface plasmon field-enhanced fluorescence spectroscopy was employed for real-time monitoring of DNA hybridizations to surface bound PNA. Probes with three different strand lengths were immobilized by self-assembly on the sensor surface. By introducing Cy5-labeled DNA targets the affinity constants, K(A)=k(on) (association)/k(off) (dissociation), were determined for fully complementary (MM0) as well as for single base mismatched (MM1) duplexes. Furthermore, the thermal stability of each duplex was determined by measuring melting curves in solution which was then compared to the kinetic and affinity parameters determined for the surface hybridization reactions. The results indicate that ions do not play a significant role for the PNA/DNA hybridization kinetics at surfaces. However, changes in the configuration of the PNA/DNA duplex due to the ionic strength variations influence the fluorescence yield drastically.
肽核酸（PNA）是寡核苷酸的模拟物，含有中性类似肽的骨架，能够与互补的 DNA 靶标高亲和力和选择性地结合。为了研究缓冲溶液离子强度的影响，在不同盐溶液中进行了 PNAs 作为（捕获）探针和 DNA 作为靶标寡核苷酸的杂交实验。表面等离子体场增强荧光光谱法用于实时监测表面结合的 PNA 与 DNA 的杂交。通过自组装将具有三种不同链长的探针固定在传感器表面。通过引入 Cy5 标记的 DNA 靶标，确定了完全互补（MM0）和单碱基错配（MM1）双链体的亲和力常数 K(A)=k(on)（结合）/k(off)（解离）。此外，通过测量溶液中的熔解曲线来确定每个双链体的热稳定性，然后将其与表面杂交反应确定的动力学和亲和力参数进行比较。结果表明，离子在表面的 PNA/DNA 杂交动力学中不起重要作用。然而，由于离子强度变化导致 PNA/DNA 双链体的构象发生变化，荧光产率会急剧变化。
