Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy.
Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy.
Biochim Biophys Acta Gen Subj. 2018 Jun;1862(6):1276-1282. doi: 10.1016/j.bbagen.2018.03.002. Epub 2018 Mar 8.
G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens.
SPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures.
The use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability.
Optimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells.
The heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface.
G-四链体（G4s）是在富含鸟嘌呤的序列中形成的核酸二级结构。抗 G4 抗体是直接研究细胞中 G4s 的工具。表面等离子体共振（SPR）是一种高灵敏度的技术，适用于评估生物分子之间的亲和力。我们旨在改善抗 G4 抗体在 SPR 传感器芯片上的取向，以优化对结合抗原的检测。
SPR 用于表征抗 G4 抗体与 G4 和非 G4 寡核苷酸的相互作用。葡聚糖功能化的传感器芯片用于共价偶联和捕获程序。
使用两种领先的分子来定向感兴趣的抗体，可将其活性从完全无功能提高到 65%。因此，可以用高灵敏度和可靠性评估抗 G4 抗体对 G4 结构的特异性。
SPR 生物传感中固定化方案的优化使我们能够以比其他体外测定（如 ELISA）更高的灵敏度确定抗 G4 抗体对 G4 抗原的亲和力和特异性。抗 G4 抗体的特异性是未来直接在细胞中监测 G4s 时利用这种抗体的基本假设。
胺偶联固定配体的异质取向是一个普遍的问题，通常会导致分子部分或完全失活。在这里，我们描述了一种改善配体取向的新策略：从两个方向驱动它。这个原则几乎可以应用于每一种在标准偶联到 SPR 芯片表面后失去活性或固定化不良的分子。
