Two-Dimensional Combinatorial Screening of a Bacterial rRNA A-Site-like Motif Library: Defining Privileged Asymmetric Internal Loops That Bind Aminoglycosides

Abstract

RNAs have diverse structures that are important for biological function. These structures include bulges and internal loops that can form tertiary contacts or serve as ligand binding sites. The most commonly exploited RNA drug target for small molecule intervention is the bacterial ribosome, more specifically the ribosomal RNA aminoacyl-tRNA site (rRNA A-site) which is a major target for the aminoglycoside class of antibiotics. The bacterial A-site is composed of a 1×1 nucleotide all-U internal loop and a 2×1 nucleotide all-A internal loop separated by a single GC base pair. Therefore, we probed the molecular recognition of a small library of four aminoglycosides for binding a 16384-member bacterial rRNA A-site-like internal loop library using Two-Dimensional Combinatorial Screening (2DCS). 2DCS is a microarray-based method that probes RNA and chemical spaces simultaneously. These studies sought to determine if aminoglycosides select their therapeutic target if given a choice of binding all possible internal loops derived from an A-site-like library. Results show that the bacterial rRNA A-site was not selected by any aminoglycoside. Analyses of selected sequences using the RNA Privileged Space Predictor (RNA-PSP) program show that each aminoglycoside preferentially binds different types of internal loops. For three of the aminoglycosides, 6″-azido-kanamycin A, 5-O-(2-azidoethyl) neamine, and 6″-azido-tobramycin, the selected internal loops bind with ~10-fold higher affinity than the bacterial rRNA A-site. The internal loops selected to bind 5″-azido-neomycin B bind with similar affinity as the therapeutic target. Selected internal loops that are unique for each aminoglycoside have dissociation constants ranging from 25 to 270 nM and are specific for the aminoglycoside they were selected to bind compared to the other arrayed aminoglycosides. These studies further establish a database of RNA motifs that are recognized by small molecules that could be used to enable the rational and modular design of small molecules targeting RNA.Most cellular RNAs are single stranded and fold back onto themselves to minimize their free energy. This provides RNA with structural diversity, forming a variety of motifs such as bulges, internal loops, hairpin loops, and multibranch loops. These individual motifs in RNA often dictate the function of the larger biomolecule. For example, a hairpin loop and an internal loop form the tetraloop receptor in group I and group II introns (1-3). Deletion of these structures impairs self-splicing (1-3). Riboswitches are another functionally important class of RNAs whose structure dictates function. These RNAs alter their structures in response to the concentration of metabolites to either stimulate or repress translation of mRNAs that contain *Author to whom correspondence should be addressed: mddisney@buffalo.edu; . Supporting Information Supporting information containing the entire selected sequences, the overlap analysis of these sequences, and the output of RNA-PSP analysis of the sel...

Defining the RNA Internal Loops Preferred by Benzimidazole Derivatives via 2D Combinatorial Screening and Computational Analysis

“…9,10,13,14 These studies found that selected interactions can occur with nanomolar K d 's to internal loops 9,10,13 and low micromolar K d 's to hairpin loops. 11,14 Aminoglycosides have a large number of amines and are therefore highly cationic ligands.…”

Identifying the preferred RNA motifs and chemotypes that interact by probing millions of combinations

“…Excellent signal was still observed for all compounds. Subsequently, compounds PL-1 , HL-1 , HL-2 , and DL-1 were subjected to microarray-based selection using the oligonucleotides competitors described above (Figure 2) 20,21,48 . Three rounds of selection were completed prior to sequencing the RNA motifs that bind each ligand.…”

“…Microarrays were constructed as previously described 20,21,48–56 . Compounds that were identified as hits from the dye-displacement assay were immobilized onto aldehyde-functionalized microarray via a reductive amination reaction.…”

Molecular Recognition of 6′-N-5-Hexynoate Kanamycin A and RNA 1x1 Internal Loops Containing CA Mismatches

“…Similar specificities have been observed with aminoglycosides binding to RNA internal and hairpin loops when the RNAs have been selected to bind to aminoglycosides via 2DCS. (30, 48-51) Other studies have suggested that aminoglycoside recognition is shape-dependent. (47) The results presented here suggest that high affinity binding is also sequence dependent.…”