A click chemistry-based microRNA maturation assay optimized for high-throughput screening

Lorenz, Daniel A.; Garner, Amanda L.

doi:10.1039/c6cc02894b

Cited by 30 publications

(47 citation statements)

References 19 publications

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“…For the click chemistry detection step, we chose our second-generation approach, which utilizes inverse-electron demand Diels–Alder (IEDDA) chemistry, and HRP was labeled with trans-cyclooctene (TCO). 20 We have previously demonstrated that, because of its kinetic superiority, replacing our first-generation copper-catalyzed alkyne–azide cycloaddition (CuAAC) click chemistry detection step 21,22 with IEDDA allowed us to develop a technology with improved sensitivity and reproducibility, enabling automated HTS. 20 Similar results were observed using CuAAC with PPI cat-ELCCA (Figure S3).…”

Section: Resultsmentioning

confidence: 99%

High-Throughput Chemical Probing of Full-Length Protein–Protein Interactions

et al. 2017

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Human biology is regulated by a complex network of protein–protein interactions (PPIs), and disruption of this network has been implicated in many diseases. However, the targeting of PPIs remains a challenging area for chemical probe and drug discovery. Although many methodologies have been put forth to facilitate these efforts, new technologies are still needed. Current biochemical assays for PPIs are typically limited to motif–domain and domain–domain interactions, and assays that will enable the screening of full-length protein systems, which are more biologically relevant, are sparse. To overcome this barrier, we have developed a new assay technology, “PPI catalytic enzyme-linked click chemistry assay” or PPI cat-ELCCA, which utilizes click chemistry to afford catalytic signal amplification. To validate this approach, we have applied PPI cat-ELCCA to the eIF4E–4E-BP1 and eIF4E–eIF4G PPIs, key regulators of cap-dependent mRNA translation. Using these examples, we have demonstrated that PPI cat-ELCCA is amenable to full-length proteins, large (>200 kDa) and small (∼12 kDa), and is readily adaptable to automated high-throughput screening. Thus, PPI cat-ELCCA represents a powerful new tool in the toolbox of assays available to scientists interested in the targeting of disease-relevant PPIs.

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Section: Resultsmentioning

confidence: 99%

High-Throughput Chemical Probing of Full-Length Protein–Protein Interactions

et al. 2017

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“…The scheme for cat-ELCCA for Dicer-mediated pre-miRNA maturation is shown in Fig. 2 (Garner, 2018a;Lorenz et al, 2015;Lorenz & Garner, 2016. In brief, a chemically synthesized, trans-cyclooctene (TCO)-labeled pre-miRNA substrate is first immobilized into the wells of a 384-well streptavidin-coated microtiter plate.…”

Section: Cat-elcca For Dicer-mediated Pre-mirna Maturationmentioning

confidence: 99%

“…To facilitate the discovery of new chemical entities for targeting RNAs, we have developed high-throughput screening (HTS) technology termed catalytic enzyme-linked click chemistry assay, or cat-ELCCA, which is a robust screening platform that does not suffer from compound interference (Garner, 2018a;Lorenz & Garner, 2016Lorenz, Song, & Garner, 2015;Lorenz, Vander Roest, Larsen, & Garner, 2018). Key advantages of cat-ELCCA include its increased sensitivity due to catalytic signal amplification, negligible compound interference in comparison to traditional fluorescence-based assays due to added washing steps, and HTS applicability with Z' factors >0.6 using automated liquid handling (Garner, 2018a;Garner & Janda, 2010, 2011Lorenz & Garner, 2016Lorenz, Kaur, et al, 2018;Lorenz et al, 2015;Lorenz, Vander Roest, et al, 2018;Song, Menon, Mitchell, Johnson, & Garner, 2017;Zhang, Chung, & Oldenburg, 1999). As proof of concept for RNA-targeted probe discovery, we applied cat-ELCCA toward the goal of identifying selective and RNA-binding inhibitors of Dicer-mediated microRNA (miRNA or miR) maturation ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…As proof of concept for RNA-targeted probe discovery, we applied cat-ELCCA toward the goal of identifying selective and RNA-binding inhibitors of Dicer-mediated microRNA (miRNA or miR) maturation ( Fig. 1) (Garner, 2018a;Lorenz & Garner, 2016Lorenz et al, 2015;Lorenz, Vander Roest, et al, 2018). Using this approach, we have recently completed a HTS of commercial small molecules and NPEs to identify compounds and areas of chemical space capable of selectively inhibiting the processing of oncogenic pre-miR-21 (Lorenz, Vander Roest, et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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A click chemistry assay to identify natural product ligands for pre-microRNAs

Garner

Lorenz

Gallagher

2019

Methods in Enzymology

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Despite the great diversity of structure and function and relevance to human health, RNA remains an underexploited area of drug discovery. A major bottleneck toward this goal has been the identification of probes and drug leads that are specific for select RNAs and methods that will facilitate such discovery efforts. Our laboratory has recently developed an innovative approach for assaying RNA-small molecule interactions, catalytic enzyme-linked click chemistry assay or cat-ELCCA, which is a functional assay that takes advantage of the power of catalytic signal amplification combined with the selectivity and bioorthogonality of click chemistry. Importantly, through application of this platform assay technology to the challenging problem of identifying selective inhibitors of pre-microRNA maturation, we identified natural products as a potential source of such compounds. Herein we describe this methodology in addition to the downstream pipeline toward the discovery of natural product ligands for premicroRNAs. Through cat-ELCCA, our goal is to discover novel ligands to facilitate our investigation of RNA recognition by small molecules.

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“…42 Over the past few years, the Garner laboratory has developed high-throughput screening technology for the discovery of small molecule modulators of RNA biology, including RPIs. [43][44][45][46] Most recently, we have developed a click chemistry-mediated complementation assay, a homogeneous platform in which signal is dependent upon RPI-driven protein complementation of a split luciferase engineered from NanoLuc (NanoLuc Binary Technology; NanoBiT), allowing for catalytic signal amplification upon detection of full-length RPIs. 47 As our previously developed assay platforms were biochemical in nature, to further advance our ability to study and manipulate RNA biology, we became interested in the development of a live-cell assay for RPIs.…”

Section: Introductionmentioning

confidence: 99%

A Live-Cell Assay for the Detection of pre-microRNA-Protein Interactions

Lorenz

Garner

2020

Preprint

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Recent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of both coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry-and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-ofconcept, we have utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.

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A click chemistry-based microRNA maturation assay optimized for high-throughput screening

Cited by 30 publications

References 19 publications

High-Throughput Chemical Probing of Full-Length Protein–Protein Interactions

High-Throughput Chemical Probing of Full-Length Protein–Protein Interactions

A click chemistry assay to identify natural product ligands for pre-microRNAs

A Live-Cell Assay for the Detection of pre-microRNA-Protein Interactions

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