聚电解质驱动DNA纳米机器的构建及其应用初探

Recently, a nanometer-scaled DNA machine，which can move and transform based on assembled structures has become a powerful tool in DNA nanotechnology fields. For most of these machines constructed so far, oligonucleotides have been generally used as the fuel. However, the switch frequency is limited by the hybridization kinetics of complementary strands; it needs for high concentration of DNA oligonucleotide to retain the machine’s dynamic action; a DNA duplex waste is produced in each cycle, accumulation of the waste products leads to a progressive loss of operating efficiency. To overcome these weaknesses, a new strategy is therefore required for the development of DNA nanotechnology. Herein we focuse on a kind of specific DNA - aptamer, which can form a hairpin structure with a self-complementary sequence in the loop and the stem. The purpose of this study was to utilize aptamer as the machine body and constructed novel nanomachines driven by polyelectrolytes, which could be instructed repeatedly with high response and without any additional DNA fuel. Cationic comb-type copolymer and anionic polymer are designed and synthesized in this work. The cationic polymer stabilizes the bimolecular duplex more effectively, and thus hairpin aptamer sequence easily transforms to bimolecular duplex in present of cationic polymer. We also found, that the anionic polymer can dissociate the cationic copolymer from the biomolecular duplex. The duplex is then changed back to original hairpin and the cycle can be repeated. Our aim is to construct a aptamer-type DNA nanomachine as smart tumor-targeted delivery system, which can activate antitumor activity of the recombinant proteins only at tumor site. The operating principle is that the aptamer can switch off protein activity to avoid nonspecific interactions and side effects at normal tissues. In the tumor site, the active protein can be released quickly and efficiently owing to the induced structural change. Using this smart device, we can control the anti-tumor activity and releasing site of the recombinant proteins..

近年来，利用DNA组装出纳米尺度下能够运动并实现能量转换的“DNA分子机器”，成为这个领域更具挑战性的前沿课题。到目前为止，绝大多数的DNA分子机器基于链交换反应，将DNA作为驱动机器运行的“燃料”。然而链交换反应速度较慢，需要较高浓度的DNA才能驱动机器的运行，每次循环结束后的残留物也会导致马达效率和寿命的迅速衰减。因此需要构建新型的DNA分子机器。我们采用聚电解质作为“燃料”，阳离子聚合物与DNA，以及阳离子聚合物与阴离子聚合物之间有很强的结合力，因此，我们以梳型阳离子共聚物和阴离子聚合物作为DNA分子机器的“燃料”驱动其快速、有效运转。我们选取结合抗肿瘤活性功能蛋白的适配体作为目标DNA，在肿瘤部位注射阳离子共聚物，发卡型适配体转为直链型二聚体，抗肿瘤活性蛋白释放，这就实现了功能蛋白的靶向定位，为DNA纳米机器的抗肿瘤应用提供了可能。

利用DNA组装出纳米尺度下的分子机器，对我们认识DNA的构型转换规律、生物分子的相互作用与调控机制有着重要意义。DNA分子机器运转的基础是DNA构型间的可控转换。我们合成出一种含有多糖支链的梳型阳离子共聚物，其可以促进DNA的杂化及链交换反应，并且提高DNA多级结构的稳定性，从而驱动DNA纳米机器实现快速、有效的运转。在此基础上，我们筛选出结合功能蛋白的适配体作为目标DNA，通过适配体结构的变化实现功能蛋白的可控和靶向释放。具体研究内容包括以下几个方面：（1）选择聚赖氨酸（PLL）作为主链，葡聚糖（Dextran）作为支链，通过还原胺化反应制备出梳型阳离子共聚物PLL-g-Dex；通过改变葡聚糖支链的含量，以调整聚合物的电位及其与DNA结合的稳定性。（2）氧化石墨烯可以吸附带荧光标记的单链DNA，此时荧光淬灭；在PLL-g-Dex的作用下，另一完全互配或部分错配的单链DNA可以与荧光标记DNA快速杂交，使其脱离氧化石墨烯；通过荧光吸光强度恢复的程度来判断目标DNA的碱基是否与靶向DNA匹配，由此构建出一种简单，快速，精确的碱基错配检测方法。（3）选取一种具有自补偿序列结构的茎冠型DNA，当PLL-g-Dex加入后，两个茎冠型DNA单体快速结合形成直链DNA二聚体；随后向体系中加入阴离子聚合物PVS，其与阳离子聚合物结合，直链型DNA二聚体恢复为茎冠型单体；如此循环往复，由于加入的聚电解质溶液以及体系中的DNA浓度都为nM浓度级，由此构建出聚电解质驱动下的DNA纳米机器。（4）利用PLL-g-Dex促进DNA结构单元的快速高效自组装，同时提高DNA多级结构的稳定性；设计一条单链S-DNA，将其通过粘性末端的杂化反应，与结构单元形成三维DNA水凝胶；我们还通过点击化学反应在交联网络结构中引入一种或多种核酸适配体，特异性结合一种或多种功能蛋白，通过加入与适配体互补的DNA单链，实现特异性功能蛋白的可控和靶向释放，从而为其在肿瘤检测与治疗方面的应用提供了理论基础。