Assessment of Molecular Interactions through Magnetic Relaxation

Abstract

Magnetic Interactions: The dissociation constant (KD) of various molecular interactions was determined using a novel competition assay with binding magnetic relaxation nanosensors (bMRs). In this assay, changes in the magnetic relaxation of an aqueous suspension of the nanosensors facilitate the fast determination of KD values using nanomolar protein concentrations.

Identification of Toxin Inhibitors Using a Magnetic Nanosensor‐Based Assay

“…In order to calculate the K D of the discovered interactions we used a previously published assay. [ 6 ] We utilized a small-molecule(SM)-carrying bMR at a concentration of 0.015 mg Fe/mL and prepared an analyzing solution that consisted of 4.5 μ L SM-carrying bMRs and 2,000 μ L of de-ionized water. Samples containing 10 μ L of different concentrations of free-SM (competing ligand .05 -500 μ M, in DI water) and 200 μ L of the SM-bMR analyzing solution (bMR nanosensor) were prepared and incubated with 10 μ L of LF (1 nM).…”

“…If a potential interaction between LF and a small molecule is identifi ed, the same bMR nanosensor is allowed to compete for binding to LF in the presence of increasing concentrations of the free small molecule in solution (competitor), allowing confi rmation of the interaction and estimation of the K D value through magnetic relaxation. [ 6 ] Our screening identifi ed 3 molecules, sulindac, naproxen and fusaric acid, as compounds that bind to LF with K D values in the micromolar range. Out of these 3 molecules, sulindac, an FDA-approved drug marketed in the USA as Clinoril ® for the treatment of pain and swelling associated with osteoarthritis was found to inhibit LF protease activity (IC 50 = 173 μ M) and reduced LF cytotoxicity (IC 50 = 31.7 μ M) to macrophages.…”

“…[1][2][3][4][5] Recently, we reported the development of an assay which uses binding magnetic relaxation (bMR) nanosensors to assess molecular interactions by measuring changes in magnetic relaxation upon ligand binding. [ 6 ] In this assay, a small molecule and a magnetic relaxation nanosensor conjugated with the same small molecule (bMR nanosensor) compete for binding to a particular target protein in solution or cellular receptor in cultured cells. The magnetic relaxation changes produced by the competitive binding produce a sigmoidal signal response from which the dissociation constant (K D ) of that particular interaction can be calculated.…”

“…Further confi rmation of this molecular interaction between LF and the small molecules on the nanoparticle was obtained by measuring the dissociation constant (K D ) using a competition magnetic relaxation assay. [ 6 ] Briefl y, the LF toxin and the bMR nanosensors were incubated with increasing amounts of the corresponding free small molecules (sulindac, naproxen or fusaric acid) as competitor. The competition of the bMR nanosensor and the increasing amounts of free molecules in solution for binding to the toxin provided us with a method to estimate the K D of the studied interactions ( Figure 1 d-f) following a previously reported method.…”

“…The competition of the bMR nanosensor and the increasing amounts of free molecules in solution for binding to the toxin provided us with a method to estimate the K D of the studied interactions ( Figure 1 d-f) following a previously reported method. [ 6 ] The corresponding Δ MR competitor values were calculated by fi rst subtracting the changes in T 2 caused by the addition of the competitor ( Δ T 2 (competitor) ) from the maximum possible change in T 2 ( Δ T 2 (max) ), which occurs in the absence of the competitor. This value is then divided by the Δ T 2 (max) in order to obtain the Δ MR competitor .…”