Dipartimento di Chimica delle Sostanze Naturali, Università di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy.
Bioconjug Chem. 2012 Mar 21;23(3):382-91. doi: 10.1021/bc200305t. Epub 2012 Feb 7.
In the present work, we report the conjugation of superparamagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleotide (ODN) able to fold into stable unimolecular guanine quadruple helix under proper ion conditions by means of its thrombin-binding aptamer (TBA) sequence. The novel modified ODN, which contained a fluorescent dU(Py) unit at 3'-end and a 12-amino-dodecyl spacer (C(12)-NH(2)) at 5' terminus, was characterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. From CD and fluorescence experiments, we verified that dU(Py) and C(12)-NH(2) incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN with the magnetite particles was performed, and the ODN-conjugated nanoparticles were studied through optical spectroscopy (UV, CD, fluorescence) and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymatic degradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in view of the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.
在本工作中，我们报告了超顺磁纳米粒子与荧光标记的寡脱氧核苷酸（ODN）的缀合，该寡脱氧核苷酸能够通过其凝血酶结合适体（TBA）序列在适当的离子条件下折叠成稳定的单分子鸟嘌呤四链体。新型修饰的 ODN 在 3'末端含有荧光 dU(Py)单元，在 5'末端含有 12-氨基-十二烷基间隔物（C(12)-NH(2))，通过 ESI-MS 和光学光谱（UV、CD、荧光）进行了表征，并通过反相高效液相色谱（RP-HPLC）色谱和电泳进行了分析。通过 CD 和荧光实验，我们验证了 dU(Py)和 C(12)-NH(2)的掺入不干扰 G-四链体的构象稳定性。随后，进行了带有芘标记的 ODN 与磁铁矿颗粒的缀合，通过光学光谱（UV、CD、荧光）以及酶和化学测定研究了 ODN 缀合的纳米颗粒。我们发现纳米颗粒增强了 TBA ODN 对酶降解的稳定性。最后，我们评估了固定在磁性分离器上的 TBA 缀合纳米颗粒的数量，以期将纳米系统用于从复杂混合物中磁性捕获凝血酶。
