Yamayoshi Asako, Miyoshi Daisuke, Zouzumi Yu-Ki, Matsuyama Yohei, Ariyoshi Jumpei, Shimada Naohiko, Murakami Akira, Wada Takehiko, Maruyama Atsushi
The Hakubi Center for Advanced Research, Kyoto University , Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan.
Department of Chemistry, Graduate School of Science, Kyoto University , Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
J Phys Chem B. 2017 Apr 27;121(16):4015-4022. doi: 10.1021/acs.jpcb.7b01926. Epub 2017 Apr 10.
DNA sequences capable of forming triplexes induce DNA double-strand breaks that have attracted attention in genome editing technologies (e.g., CRISPR/Cas9 system, TALEN, and ZFN). Therefore, novel functional tools that stabilize triplex DNA structures must be further investigated to spark renewed interest. In this study, we investigated the unique character of cationic comb-type copolymers for the selective stabilization of triplex DNA. The melting temperature (T) of triplex DNA increased from 24.5 to 73.0 °C (ΔT = 48.5 °C) by the addition of poly(allylamine)-graft-dextran (PAA-g-Dex) under physiological conditions (at pH 7.0), while PAA-g-Dex did not stabilize but rather destabilized the DNA duplex. On the other hand, poly(l-lysine)-graft-dextran (PLL-g-Dex) stabilized both the duplex and triplex structures at pH 7.0. Thermodynamic parameters evaluated by isothermal titration calorimetry (ITC) revealed that the binding constant (K) for the intermolecular triplex formation in the presence of PAA-g-Dex was 1.1 × 10 M at 25 °C which is more than 10 times larger than that in the presence of PLL-g-Dex (8.6 × 10 M). Stabilizing activity and selectivity of cationic copolymers toward DNA assemblies were successfully controlled by selecting appropriate backbone structures of the copolymer. Various functional molecules that stabilize DNA duplexes have been developed and used in biological research. However, there are few cationic polymers that stabilize triplex DNA selectively. This study indicates that PAA-g-Dex has great potential to regulate the biological activities of triplex DNA.
能够形成三链体的DNA序列会诱导DNA双链断裂，这在基因组编辑技术（如CRISPR/Cas9系统、转录激活因子样效应物核酸酶和锌指核酸酶）中引起了关注。因此，必须进一步研究能够稳定三链体DNA结构的新型功能工具，以重新激发人们的兴趣。在本研究中，我们研究了阳离子梳型共聚物对三链体DNA的选择性稳定作用的独特特性。在生理条件下（pH 7.0），通过添加聚烯丙胺接枝葡聚糖（PAA-g-Dex），三链体DNA的解链温度（Tm）从24.5℃升高到73.0℃（ΔTm = 48.5℃），而PAA-g-Dex并没有稳定DNA双链体，反而使其不稳定。另一方面，聚L-赖氨酸接枝葡聚糖（PLL-g-Dex）在pH 7.0时稳定了双链体和三链体结构。通过等温滴定量热法（ITC）评估的热力学参数表明，在25℃下，存在PAA-g-Dex时分子间三链体形成的结合常数（K）为1.1×10⁶ M，比存在PLL-g-Dex时（8.6×10⁵ M）大10倍以上。通过选择共聚物的合适主链结构，成功地控制了阳离子共聚物对DNA组装体的稳定活性和选择性。已经开发出各种稳定DNA双链体的功能分子并将其用于生物学研究。然而，很少有阳离子聚合物能选择性地稳定三链体DNA。本研究表明，PAA-g-Dex在调节三链体DNA的生物学活性方面具有巨大潜力。
