Single-molecule study of the CUG repeat–MBNL1 interaction and its inhibition by small molecules

Abstract

Effective drug discovery and optimization can be accelerated by techniques capable of deconvoluting the complexities often present in targeted biological systems. We report a single-molecule approach to study the binding of an alternative splicing regulator, muscleblind-like 1 protein (MBNL1), to (CUG)n = 4,6 and the effect of small molecules on this interaction. Expanded CUG repeats (CUGexp) are the causative agent of myotonic dystrophy type 1 by sequestering MBNL1. MBNL1 is able to bind to the (CUG)n–inhibitor complex, indicating that the inhibition is not a straightforward competitive process. A simple ligand, highly selective for CUGexp, was used to design a new dimeric ligand that binds to (CUG)n almost 50-fold more tightly and is more effective in destabilizing MBNL1–(CUG)4. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions.

Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy

“…At the beginning of imaging, use 5 seconds excitation with 640 nm laser, then switch the excitation source to 532 nm laser for the rest of the recording. In this scheme, the MatLab program for extraction of the fluorescence trajectories only needs to consider the first 5 seconds of the recording to map the possible locations of surface-tethered molecules, aiding in the selection of trajectories (Boehm et al, 2016, Haghighat Jahromi et al, 2013). When the unlabeled neutravidin is used for surface-tethering, the MatLab code needs to be set to scan the entire movie in Cy3 channel to identify the locations where the Cy3 signal associated with the binding of the freely diffusing Cy3-labeled ligand appears.…”

“…More complex binding patterns can be observed if the surface-tethered molecule contains multiple binding sites for the fluorescently-labeled binding partner(s), which may bind independently or oligomerize upon binding to the surface-tethered substarte. An example of the interaction involving multiple independent binding sites can be found in a study examining the interaction of muscleblind-like 1 protein (MBNL1) with RNA molecules containing CUG repeats (Haghighat Jahromi et al, 2013). The protein bound the surface-tethered (CUG) 4 construct with 1:1 stoichiometry.…”

“…Even in its simplest one-color version, TIRFM-based single-molecule co-localization provides a suite of powerful approaches to develop a comprehensive understanding of the inhibition mechanism and the kinetics of the macromolecular interactions in the presence of an inhibitor (Haghighat Jahromi et al, 2013). In this section we provide a guide on how to design and carry this type of analysis.…”

“…In this section we provide a guide on how to design and carry this type of analysis. We will use a study of the non-competitive inhibition of the interaction between muscleblind-like 1 protein (MBNL1) and the CUG repeat containing RNA (Haghighat Jahromi et al, 2013), but the overall approach can be generally applied to competitive and non-competitive inhibitors, as well as the allosteric effectors of protein-protein and protein nucleic acid interactions. The major advantage of this approach is that it is model-independent, i.e.…”

“…Global analysis of these trajectories allows for accurate determination of the kinetic rate constants and interaction affinities (Wang et al, 2009, Elenko et al, 2009, Huppa et al, 2010, Joshi et al, 2012, Haghighat Jahromi et al, 2013, Chen et al, 2014), helps to reveal populations of conformers with different binding or catalytic properties (Amrute-Nayak et al, 2014, Chen et al, 2016, Honda et al, 2014), allows the investigator to distinguish different orders of complex assembly, and provides the tools to infer the mechanisms of competition or cooperation in the assembly of the higher order complexes (Boehm, 2016, Hoskins et al, 2016). With the advent of commercially available TIRFM systems, the methodology is becoming less arcane and hopefully will become a routine technique in biophysics and biochemistry laboratories.…”