A cross-linking approach to map small molecule-RNA binding sites in cells

Velagapudi, Sai Pradeep; Li, Yue; Disney, Matthew D.

doi:10.1016/j.bmcl.2019.04.001

Cited by 34 publications

(42 citation statements)

References 18 publications

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“…Next, we determined whether we could map the binding site(s) of 2 using Chem-CLIP-Map ( 9 , 23 ). In this approach, the RNA is cross-linked to the Chem-CLIP probe, followed by analysis by primer extension, or a reverse-transcription (RT) reaction.…”

Section: Resultsmentioning

confidence: 99%

“…In the present work, we integrated FFFs with tools for studying transcriptome-wide RNA ligandability—specifically, chemical cross-linking and isolation by pull-down (Chem-CLIP), which enables the identification of RNA targets and corresponding binding sites ( 23 , 24 ). This integrated approach, dubbed Chem-CLIP fragment mapping (Chem-CLIP-Frag-Map), uses libraries of FFFs ( 21 ) to identify small molecules that engage specific RNA targets and maps their binding sites using Chem-CLIP-Map ( 23 ). We applied this strategy to develop a potent and selective bioactive ligand targeting the precursor to microRNA-21 (pre-miR-21), a ncRNA linked to cancers ( 25 ) and other indications ( 26 – 28 ) ( Fig.…”

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confidence: 99%

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A general fragment-based approach to identify and optimize bioactive ligands targeting RNA

Suresh

Zhang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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RNAs have important functions that are dictated by their structure. Indeed, small molecules that interact with RNA structures can perturb function, serving as chemical probes and lead medicines. Here we describe the development of a fragment-based approach to discover and optimize bioactive small molecules targeting RNA. We extended the target validation method chemical cross-linking and isolation by pull-down (Chem-CLIP) to identify and map the binding sites of low molecular weight fragments that engage RNA or Chem-CLIP fragment mapping (Chem-CLIP-Frag-Map). Using Chem-CLIP-Frag-Map, we identified several fragments that bind the precursor to oncogenic microRNA-21 (pre-miR-21). Assembly of these fragments provided a specific bioactive compound with improved potency that inhibits pre-miR-21 processing, reducing mature miR-21 levels. The compound exerted selective effects on the transcriptome and selectively mitigated a miR-21–associated invasive phenotype in triple-negative breast cancer cells. The Chem-CLIP-Frag-Map approach should prove general to expedite the identification and optimization of small molecules that bind RNA targets.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

A general fragment-based approach to identify and optimize bioactive ligands targeting RNA

Suresh

Zhang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, pulled down RNA can be reverse transcribed with a gene-specific RT primer, followed by addition of adaptors and amplification via RT-PCR. Chem-CLIP-Map-Seq was validated with Targaprimir-96, demonstrating that the reaction site for the Chem-CLIP probe is proximal to the SMIRNA binding site and in accordance with Inforna design and modeling (Velagapudi et al, 2019).…”

Section: Chemical Cross-linking and Isolation By Pull-down (Chem-clip)mentioning

confidence: 72%

“…For other RNA targets, RT-PCR, including RNA-seq analysis, can be used to define cellular binding sites. In these experiments, the site that the probe reacts with in an RNA target can be detected as an "RT stop" (Sexton, Wang, Rutenberg-Schoenberg, & Simon, 2017;Velagapudi, Li, & Disney, 2019). Thus, pulled down RNA can be reverse transcribed with a gene-specific RT primer, followed by addition of adaptors and amplification via RT-PCR.…”

Section: Chemical Cross-linking and Isolation By Pull-down (Chem-clip)mentioning

confidence: 99%

Identifying and validating small molecules interacting with RNA (SMIRNAs)

Disney

Velagapudi

et al. 2019

Methods in Enzymology

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High throughput sequencing has revolutionized our ability to identify aberrant RNA expression and mutations that cause or contribute to disease. These data can be used directly to design oligonucleotide-based modalities using Watson-Crick pairing to target unstructured regions in an RNA. A complementary, although more difficult, strategy to deactivate a malfunctioning RNA is to target highly structured regions with small molecules. Indeed, RNA structures are directly causative of disease. Herein, we discuss emerging strategies to design high affinity, selective, bioactive ligands targeting RNA, or small molecules interacting with RNA (SMIRNAs), and target validation and profiling methods. An experimental foundation is required for a lead identification strategy for RNA structures, constructed from a library-vs.-library screen that probes vast libraries of small molecules for binding RNA three dimensional folds. Dubbed 2-dimensional combinatorial screening (2DCS), the resulting data can be mined against transcriptomes or the composite of RNAs that are produced in an organism to define folded RNA structures that can be targeted. By applying SMIRNAs to cells and using target validation tools such as Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP) and Small Molecule Nucleic Acid Profiling by Cleavage Applied to RNA (RiboSNAP), all targets engaged in cells can be defined, along with rules for molecular recognition to affect RNA biology. This chapter will describe lessons learned in applying these approaches in vitro, in cells, and in pre-clinical animal models of disease, enabling SMIRNAs to capture opportunities in chemical biology.

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“…Other validation methods include surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), cryoEM, and chemical probing (for review, see [ 19 ]). In reverse, Chemical Cross-Linking and Isolation by Pull-down to Map Small Molecule-RNA Binding Sites (Chem-CLIP-Map-Seq) can be used to confirm the specificity of RNA-small molecule interaction [ 60 ]. Briefly, a small molecule conjugated to beads is crosslinked to its RNA targets, which are then identified through RNA-seq, therefore providing information about the specificity of the interaction between the small molecule and the RNA of interest.…”

Section: Discovery and Validation Of Small Molecules Targeting Ncrnasmentioning

confidence: 99%

Small Molecule Drugs Targeting Non-Coding RNAs as Treatments for Alzheimer’s Disease and Related Dementias

Nguyen

Chau

Krichevsky

2021

Genes

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Despite the enormous burden of Alzheimer’s disease and related dementias (ADRD) on patients, caregivers, and society, only a few treatments with limited efficacy are currently available. While drug development conventionally focuses on disease-associated proteins, RNA has recently been shown to be druggable for therapeutic purposes as well. Approximately 70% of the human genome is transcribed into non-protein-coding RNAs (ncRNAs) such as microRNAs, long ncRNAs, and circular RNAs, which can adopt diverse structures and cellular functions. Many ncRNAs are specifically enriched in the central nervous system, and their dysregulation is implicated in ADRD pathogenesis, making them attractive therapeutic targets. In this review, we first detail why targeting ncRNAs with small molecules is a promising therapeutic strategy for ADRD. We then outline the process from discovery to validation of small molecules targeting ncRNAs in preclinical studies, with special emphasis on primary high-throughput screens for identifying lead compounds. Screening strategies for specific ncRNAs will also be included as examples. Key challenges—including selecting appropriate ncRNA targets, lack of specificity of small molecules, and general low success rate of neurological drugs and how they may be overcome—will be discussed throughout the review.

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A cross-linking approach to map small molecule-RNA binding sites in cells

Cited by 34 publications

References 18 publications

A general fragment-based approach to identify and optimize bioactive ligands targeting RNA

A general fragment-based approach to identify and optimize bioactive ligands targeting RNA

Identifying and validating small molecules interacting with RNA (SMIRNAs)

Small Molecule Drugs Targeting Non-Coding RNAs as Treatments for Alzheimer’s Disease and Related Dementias

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