Abstract
BackgroundG-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. MethodsSPR 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. ResultsThe 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. ConclusionsOptimization 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. General significanceThe 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.
Highlights
G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures [1,2,3] that form in guanine (G)-rich DNA and RNA sequences when Gs arrange into stacked planar quartets coordinated by Hoogsteen-type hydrogen bonds and stabilized by metal cations such as K+ and Na+ [4]
Several advantages are offered by Surface Plasmon Resonance (SPR) biosensing: data are acquired in real-time, there is no need for sample labeling and the extremely high sensitivity allows to minimize sample volumes [20]
We here aimed at measuring by SPR biosensing the kinetic and thermodynamic binding affinities of a series of G4s to the anti-G4 monoclonal antibody 1H6 [1]
Summary
G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures [1,2,3] that form in guanine (G)-rich DNA and RNA sequences when Gs arrange into stacked planar quartets coordinated by Hoogsteen-type hydrogen bonds and stabilized by metal cations such as K+ and Na+ [4]. Given the high polymorphism of G4 structures in terms of strand stoichiometry, orientation and topology, and loop length, sequence and position [16,17] the possibility to confirm G4 folding by investigating and characterizing G4 binding to anti G4-antibody is a key benefit. To this aim, Surface Plasmon Resonance (SPR) is a powerful technology: it combines cutting-edge microfluidic and optic implementations to measure biomolecules binding and kinetics [18]. 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
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