Kim W J, Ishihara T, Akaike T, Maruyama A
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori, Yokohama 226-8501, Japan.
Nucleic Acids Res Suppl. 2001(1):151-2. doi: 10.1093/nass/1.1.151.
In the previous study (Chem. Eur. J., 7, 176 (2001)) we demonstrated that the comb-type polycationic copolymer (PLL-g-Dex) which is composed of poly (L-lysine) backbone and dextran graft chains expedited the DNA strand exchange reaction. In this study, fluorescence resonance energy transfer (FRET) was employed to explore the copolymer-mediated DNA strand exchange with higher time-resolution. To initiate strand exchange reaction the duplex prepared from 3'-fluorescein isothiocyanate (FITC)--and 5'-carboxytetramethylrhodamine(TAMRA)-labeled complementary DNAs was added to its non-labeled complementary single strand. DNA strand exchange was monitored by observing the recovery of the FITC quenching. More than 20,000 times increase in strand exchange rate at 37 degrees C by the copolymer was estimated. To investigate the accelerating mechanisms of the copolymer, the same reactions but at various ionic strenghts were studied. With increasing ionic strengths the strand exchange rate in the absence of the copolymer increased, suggesting that ionic repulsion among DNAs is unfavorable for the strand exchange to occur. Hence, alleviation of the electrostatic repulsion through interpolyelectrolyte complex formation is probably a role of the copolymer for accelerating the strand exchange reaction.
在之前的研究中（《欧洲化学杂志》，第7卷，第176页（2001年）），我们证明了由聚（L-赖氨酸）主链和葡聚糖接枝链组成的梳型聚阳离子共聚物（PLL-g-Dex）加速了DNA链交换反应。在本研究中，采用荧光共振能量转移（FRET）以更高的时间分辨率探索共聚物介导的DNA链交换。为引发链交换反应，将由3'-异硫氰酸荧光素（FITC）和5'-羧基四甲基罗丹明（TAMRA）标记的互补DNA制备的双链体加入其未标记的互补单链中。通过观察FITC淬灭的恢复来监测DNA链交换。据估计，该共聚物在37℃下使链交换速率提高了20,000倍以上。为研究共聚物的加速机制，研究了在不同离子强度下的相同反应。随着离子强度增加，在没有共聚物的情况下链交换速率增加，这表明DNA之间的离子排斥不利于链交换的发生。因此，通过形成聚电解质络合物减轻静电排斥可能是共聚物加速链交换反应的一个作用。
