Native Electrospray Ionization Mass Spectrometry Reveals Multiple Facets of Aptamer–Ligand Interactions: From Mechanism to Binding Constants

Abstract

Aptamers are oligonucleotide receptors obtained through an iterative selection process from random-sequence libraries. Though many aptamers for a broad range of targets with high affinity and selectivity have been generated, a lack of high-resolution structural data and the limitations of currently available biophysical tools greatly impede understanding of the mechanisms of aptamer-ligand interactions. Here we demonstrate that an approach based on native electrospray ionization mass spectrometry (ESI-MS) can be successfully applied to characterize aptamer-ligand complexes in all details. We studied an adenosine-binding aptamer (ABA), a l-argininamide-binding aptamer (LABA), and a cocaine-binding aptamer (CBA) and their noncovalent interactions with ligands by native ESI-MS and complemented these measurements by ion mobility spectrometry (IMS), isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. The ligand selectivity of the aptamers and the respective complex stoichiometry could be determined by the native ESI-MS approach. The ESI-MS data can also help refining the binding model for aptamer-ligand complexes and deliver accurate aptamer-ligand binding affinities for specific and nonspecific binding events. For specific ligands, we found K = 69.7 μM and K = 5.3 μM for ABA (two binding sites); K = 22.04 μM for LABA; and K = 8.5 μM for CBA.

Protein secondary structure affects glycan clustering in native mass spectrometry

“…It was originally developed for higher affinity interactions, where agreement in K D to other methods was observed [5]. It also has proven invaluable for other ligand types [8,28,31]. IMMS demonstrated consistent results for HBGA B and Gb4 implying similar glycan clustering.…”

“…Clustering arises from statistical presence of free ligands within the same droplet as the free or ligand-bound proteins, which can then dry down to the protein surface upon droplet evaporation. This process becomes relevant at elevated ligand concentrations and is independent of protein molecular weight [6][7][8]. Calculations to correct for this effect are based on total peak areas per mass species within the same spectrum to ensure identical ionization conditions.…”

“…Additionally, the reported K D values from carbohydrate binding studies to P dimers are not comparable in STD NMR, native MS and isothermal titration calorimetry (ITC) [11,12,17]. Importantly, K D s obtained for active pharmaceutical agents based on different biophysical assays such as native MS, ITC, surface plasmon resonance (SPR) and circular dichroism (CD) were equivalent in numerous other cases [8,18,19]. However, these compounds showed higher binding affinities (µM range) towards the target protein and exhibited in most cases no carbohydrate-like structures [20].…”

Aptamer Selection Against Quinolones Antibiotics Using Immobilizing Free SELEX, and Studying Aptamer-Antibiotic Interaction By Spectroscopy and High-Resolution Mass Spectrometry

“…Each run can be done just in few minutes. Another valuable feature of this method is that there is no need to do any labeling, as well as the analysis by mass spectrometry does not result in a false-positive response [39][40][41] .…”

A proof of concept application of aptachain: ligand-induced self-assembly of a DNA aptamer

“…8 This aptamer is widely used in biosensor development and forms a three-way junction structure and has been shown to function when split into two or three separate strands. [8][9][10][11][12] An unusual feature of the cocaine-binding aptamer is that it is able to bind quinine 50-fold tighter than cocaine [13][14][15][16][17][18][19] and other quinine-based antimalarial compounds even tighter. 20 In the work presented here, we split the cocaine-binding aptamer in such a way that the strands overlap and can elongate in a manner resembling an oligomer-forming chain (Fig.…”