Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions

Paul, Dustin J.; Seedhouse, Steven J.; Disney, Matthew D.

doi:10.1093/nar/gkp594

Cited by 26 publications

(50 citation statements)

References 61 publications

(95 reference statements)

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“…For example, studies by Wong and co-workers showed that several aminoglycosides have only limited specificity for binding a variety of RNAs that mimic the bacterial rRNA A-site (39). In this current and other previously reported 2DCS studies (16-18, 21), specific RNA structures that bind aminoglycosides have been identified using only a single round of selection. With the bacterial rRNA A-site-like library, overlap of statistically significant trends in the 2DCS selections have helped to identify the RNA motifs that prefer to specifically bind an aminoglycoside (Figure 3).…”

Section: Resultsmentioning

confidence: 85%

“…The structures were then analyzed computationally for commonalities using the RNA P rivileged S pace P redictor (RNA-PSP) Program, version 1.1 (18). …”

Section: Methodsmentioning

confidence: 99%

“…The output of 2DCS is analyzed via the RNA-PSP program (18) to identify trends that are significant to a ≥95% confidence level. The trends for each ligand were then compared to identify those that are unique.…”

Section: Figures and Tablesmentioning

confidence: 99%

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

Tran

Disney

2010

Biochemistry

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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...

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Section: Resultsmentioning

confidence: 85%

“…The structures were then analyzed computationally for commonalities using the RNA P rivileged S pace P redictor (RNA-PSP) Program, version 1.1 (18). …”

Section: Methodsmentioning

confidence: 99%

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

Tran

Disney

2010

Biochemistry

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“…9,10,23 This approach, Two-Dimensional Combinatorial Screening (2DCS), coupled with identifying statistically significant, unique features in the selected RNAs, 11,12 afforded high affinity and selective interactions. This information was then used in drug discovery efforts to target triplet-repeating and tetra-repeating RNA transcripts that cause disease.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2011

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RNA is an important therapeutic target, however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096-member 3×3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, drug-like benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence.

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“…The authors applied a particularly clever screening methodology called 2D combinatorial screening (2DCS) [65][66][67]. In 2DCS, a small-molecules library is conjugated onto an agarose microarray surface.…”

Section: Focused Screening Approachmentioning

confidence: 99%

Small-Molecule Approaches Toward the Targeting of Oncogenic Mirnas: Roadmap for the Discovery of Rna Modulators

2016

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miRNAs are a recently discovered class of small noncoding RNAs implicated in the regulation of gene expression. The deregulation of miRNAs levels has been linked to the development of various cancers where oncogenic miRNAs are overexpressed and tumor suppressor miRNAs are underexpressed. Here we report the three main strategies developed in order to discover small-molecule drugs able to selectively interfere with oncogenic miRNAs: the high throughput screening of large libraries of compounds, the focused screening of small libraries of molecules that are known to be able to interact with RNA thus being supposed modulators of miRNAs pathway and the design of small molecules based on the secondary structure of targeted RNA and/or three-dimensional structure of enzymes involved in miRNAs pathway.

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Two-dimensional combinatorial screening and the RNA Privileged Space Predictor program efficiently identify aminoglycoside–RNA hairpin loop interactions

Cited by 26 publications

References 61 publications

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

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

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

Small-Molecule Approaches Toward the Targeting of Oncogenic Mirnas: Roadmap for the Discovery of Rna Modulators

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