Characterizing aptamer small molecule interactions with backscattering interferometry

Abstract

Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule-aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM-aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R(2) values >0.95 and an excellent signal to noise ratio at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.

Aptamer-based Field-Effect Biosensor for Tenofovir Detection

“…To compare non-specific and specific responses, a high concentration of a non-specific drug was used to show that even at such high concentrations the response of the biosensor is negligible. The aptamer used in this work has been characterized by Kammer et al 27. using backscattering interferometry with a K d value of 9 ± 1.4 nM.…”

Aptamer Grafting onto (on) and into (in) Pegylated Gold Nanoparticles: Physicochemical Characterization and In vitro Cytotoxicity Investigation in Renal Cells

“…Then, the positively charged APT in water solution shows strong electrostatic interaction with the negatively charged AuCl 4 ions leading to formation of a hybrid complex APT-AuCl 4 -(scheme 1b). The addition of PEG-COOH on the hybrid complex stabilizes it through electrostatic interaction between carbonyl and amino groups, improving the kinetics of reduction by complexation of Au ions [39] and controlling the growth process of the final nanoparticles (APT IN PEG-AuNPs). The steric arrangement of APT INPEG-Au clusters before reduction of HAuCl 4 was confirmed by UV-Vis absorption spectroscopy, TEM and Raman Spectroscopy, as reported in the following sections.…”

“…We suppose that, the ionic strength and protonation of APT onto PEG chains improve the water solubility of the APT molecules, taking advantage of the drug release from PEG-AuNPs. Indeed, a remarkable moiety of APT electrostatically binding onto dissociated complex is released, through PEG chain and diffusionphenomena [32,39].…”

An experimental check of backscattering interferometry

“…Jørgensen and colleagues did not perform sample referencing in an appropriate manner, either using the ligand (the binding partner that changes in concentration) in the absence of the receptor (the partner held at constant concentration), or with a non-binding analogue to the ligand. The need for referencing against a nonbinding control has been noted in multiple publications by us [4, 9, 10, 28] and Bornhop [11, 15] using the terms “reference” or “control” for this type of correction procedure. This measurement is not a “blank,” which is what Jørgensen and colleagues apparently used.…”