5 research outputs found
Method Development And Applications To Screening And Characterization Of Rrna-Targeting Small Molecules
A series of single ring aminoglycoside analogues was tested for binding to a model RNA representing the A site using electrospray ionization mass spectrometry (ESI-MS). Several of the synthetic analogues with low molecular weights were found to bind to the RNA with affinities comparable to the parental aminoglycoside neamine, with apparent dissociation constants in the low micromolar range. Salt dependence of the affinity constants for the single ring analogues revealed a predominantly electrostatic binding mode. Footprinting experiments revealed that one of the compounds (DHR23) has a similar binding site as the antibiotic paromomycin. DMS chemical probing results also suggest that the binding of DHR23 to the A site leads to stabilization of the stacked-in conformation of A1492 and A1493.
To aid in the ligand identification process, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. ESI-MS results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs were identified.
Furthermore, the specificity of one compound, DHR23 as well as a range of other ligands were tested for binding to a set of RNA models, as well as the modified and unmodified decoding region RNA constructs. The results show that DHR23 has preferred binding to structured RNA as compared to ssRNA, as well as a modest preference for the A-site RNA. Also our results indicate that modified nucleotides at or near the ligand-binding pocket may affect binding affinity of small molecules.
In summary, the results from this work have shown that generation of compounds based on these simplified structures in combination with FID screening may lead to selective reagents for RNA internal bulges, loops, mismatches, or other unique secondary structure elements
Cu(II)-Based Paramagnetic Probe to Study RNA–Protein Interactions by NMR
Paramagnetic NMR techniques allow
for studying three-dimensional structures of RNA–protein complexes.
In particular, paramagnetic relaxation enhancement (PRE) data can
provide valuable information about long-range distances between different
structural components. For PRE NMR experiments, oligonucleotides are
typically spin-labeled using nitroxide reagents. The current work
describes an alternative approach involving a CuÂ(II) cyclen-based
probe that can be covalently attached to an RNA strand in the vicinity
of the protein’s binding site using “click” chemistry.
The approach has been applied to study binding of HIV-1 nucleocapsid
protein 7 (NCp7) to a model RNA pentanucleotide, 5′-ACGCU-3′.
Coordination of the paramagnetic metal to glutamic acid residue of
NCp7 reduced flexibility of the probe, thus simplifying interpretation
of the PRE data. NMR experiments showed attenuation of signal intensities
from protein residues localized in proximity to the paramagnetic probe
as the result of RNA–protein interactions. The extent of the
attenuation was related to the probe’s proximity allowing us
to construct the protein’s contact surface map