Waybrant Brett, Pearce Timothy R, Kokkoli Efrosini
Department of Chemical Engineering and Materials Science, and ‡Department of Biomedical Engineering, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Langmuir. 2014 Jul 1;30(25):7465-74. doi: 10.1021/la500403v. Epub 2014 Jun 17.
Previously we identified an aptamer, named FKN-S2, which binds the cell surface protein fractalkine with high affinity and specificity. In this paper a hydrophobic dialkyl C16 tail was added to the aptamer to create an aptamer-amphiphile. We investigated how the tail and a spacer molecule of varying length and hydrophobicity, inserted between the tail and the aptamer headgroup, affect the binding, structure, and self-assembly properties of the aptamer-amphiphile. We synthesized aptamer-amphiphiles with no spacer (NoSPR), polyethylene glycol (PEG4, PEG8, PEG24), alkyl (C12 and C24), or oligonucleotide (T10 and T5: 10 and 5 thymine, and A10: 10 adenine) spacers. The addition of the tail reduced the binding affinity of the aptamer-amphiphile over 7.5-fold compared to the free aptamer. The hydrophobic alkyl spacers resulted in the greatest loss of affinity, and the hydrophilic PEG spacers improved amphiphile affinity but did not restore it to that of the free aptamer. Interestingly, oligonucleotide spacers produced the highest affinity amphiphiles. Nucleotide composition did not affect affinity, however, as the T10 and A10 spacers had equal affinity. The oligonucleotide spacer amphiphiles had the highest affinity because the oligonucleotide spacer increased the affinity of free aptamer; the FKN-S2 aptamer plus the oligonucleotide spacer had a higher affinity than the free FKN-S2 aptamer. Circular dichroism (CD) spectroscopy and thermal melting studies indicated the aptamer forms a stem-loop and intramolecular G-quadruplex, and the tail strongly stabilized the formation of the G-quadruplex in a buffer. Cryogenic transmission electron microscopy (cryo-TEM) imaging showed the aptamer-amphiphiles, independent of the spacer used, self-assembled into micelles and nanotapes, flat bilayer structures that were often twisted. Finally, liposomes functionalized with the FKN-S2 amphiphile were incubated with fractalkine expressing cells, and the amount of binding was dependent on the concentration of the amphiphile on the liposome surface.
此前我们鉴定出一种名为FKN-S2的适体，它能以高亲和力和特异性结合细胞表面蛋白趋化因子。在本文中，我们在该适体上添加了一条疏水性二烷基C16尾巴，以构建一种适体两亲分子。我们研究了这条尾巴以及插入尾巴与适体头基之间、具有不同长度和疏水性的间隔分子，是如何影响适体两亲分子的结合、结构和自组装性质的。我们合成了不含间隔分子（NoSPR）、含聚乙二醇（PEG4、PEG8、PEG24）、烷基（C12和C24）或寡核苷酸（T10和T5：10个和5个胸腺嘧啶，以及A10：10个腺嘌呤）间隔分子的适体两亲分子。与游离适体相比，尾巴的添加使适体两亲分子的结合亲和力降低了7.5倍以上。疏水性烷基间隔分子导致亲和力损失最大，而亲水性PEG间隔分子提高了两亲分子的亲和力，但未将其恢复到游离适体的水平。有趣的是，寡核苷酸间隔分子产生了亲和力最高的两亲分子。然而，核苷酸组成并不影响亲和力，因为T10和A间隔分子具有相同的亲和力。寡核苷酸间隔分子两亲分子具有最高的亲和力，是因为寡核苷酸间隔分子增加了游离适体的亲和力；FKN-S2适体加上寡核苷酸间隔分子的亲和力高于游离FKN-S2适体。圆二色（CD）光谱和热熔解研究表明，该适体形成了一个茎环和分子内G-四链体，并且在缓冲液中尾巴强烈稳定了G-四链体的形成。低温透射电子显微镜（cryo-TEM）成像显示，无论使用何种间隔分子，适体两亲分子都会自组装成胶束和纳米带，即通常扭曲的扁平双层结构。最后，用FKN-S2两亲分子功能化的脂质体与表达趋化因子的细胞一起孵育，结合量取决于脂质体表面两亲分子的浓度。
