9 research outputs found
Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface
There is a high demand for characterizing
oligonucleotide structural
changes associated with binding interactions as well as identifying
novel binders that modulate their structure and function. In this
study, second-harmonic generation (SHG) was used to study RNA and
DNA oligonucleotide conformational changes associated with ligand
binding. For this purpose, we developed an avidin-based biotin capture
surface based on a supported lipid bilayer membrane. The technique
was applied to two well-characterized aptamers, both of which undergo
conformational changes upon binding either a protein or a small molecule
ligand. In both cases, SHG was able to resolve conformational changes
in these oligonucleotides sensitively and specifically, in solution
and in real time, using nanogram amounts of material. In addition,
we developed a competition assay for the oligonucleotides between
the specific ligands and known, nonspecific binders, and we demonstrated
that intercalators and minor groove binders affect the conformation
of the DNA and RNA oligonucleotides in different ways upon binding
and subsequently block specific ligand binding in all cases. Our work
demonstrates the broad potential of SHG for studying oligonucleotides
and their conformational changes upon interaction with ligands. As
SHG offers a powerful, high-throughput screening approach, our results
here also open an important new avenue for identifying novel chemical
probes or sequence-targeted drugs that disrupt or modulate DNA or
RNA structure and function
Chemical structure of MLKL binders described in text.
<p>Chemical structure of MLKL binders described in text.</p
MSD assay monitoring RIPK3-dependent phosphorylation of the MLKL activation loop.
<p>(A) GSK872 compound that specifically inhibits RIPK3 inhibits phosphorylation of MLKL as positive control. (B) Crizotinib binding to MLKL does not impact its phosphorylation by RIPK3. (C) Cpd <b>1</b> binding to MLKL does not impact its phosphorylation by Ripk3. (D) Cpd <b>4</b> binding to MLKL does not impact its phosphorylation by RipK3. Data is normalized against a reaction in the presence of DMSO alone (100% activity).</p
Superposition of the crystal structures of MLKL pseudokinase bound to cpd 1 (green) or cpd 4 (magenta).
<p>The C-terminal domain of both structures were superimposed. Phe 350 of the GFE motif in hMLKL is highlighted to demonstrate the conformational changes induced by binding the type II cpd <b>1</b>. The catalytic lysine (K230) which is labeled by the SHG-sensitive dye is also highlighted.</p
Definition of the terms used to calculate degree of rescue or viability, based on presence or absence of compound and TNF.
<p>Definition of the terms used to calculate degree of rescue or viability, based on presence or absence of compound and TNF.</p
Necroptosis signaling pathway and selectivity of compounds in this study for one or more kinases with this pathway.
<p>(A) Necroptosis signaling pathway includes RIPK1, RIPK3 and MLKL (B) Analysis of cpd <b>1</b>% inhibition of 403 non-mutant kinases when tested at 1 μM concentration (the size of the red circle indicates a higher % inhibition). (C) Cpd <b>4</b> inhibits none of the 403 non-mutant kinases when tested at 1 μM concentration, but does inhibit pseudokinase MLKL.</p
Compound binding affinities for MLKL, RIPK1 and RIPK3, differential thermal shift values and Necroptosis Inhibition.
<p>Compound binding affinities for MLKL, RIPK1 and RIPK3, differential thermal shift values and Necroptosis Inhibition.</p
Necroptosis assay using FADD-deficient Jurkat cells measuring compound-dependent rescue from cell death.
<p>The different compounds described in text were tested for rescue in dose-response curves: (A) cpd <b>1</b> (B) cpd <b>2</b> (C) cpd <b>3</b> (D) cpd <b>4</b> (E) <b>Crizotinib</b> (F) Necrostatin (Nec1s). The blue line represents the rescue experiment from necroptosis in the presence of TNF and the yellow line represents the viability experiment in the absence of TNF.</p
Changes in SHG intensity over time upon compound binding.
<p>(A) full length (FL) MLKL and (B) pseudokinase (PK) domain SHG intensity with 10 μM compound addition. (C) Quantitation of % change overall for the kinetic curves.</p