Measuring RNA–Ligand Interactions with Microscale Thermophoresis

Moon, Michelle; Hilimire, Thomas A.; Sanders, Allix M.; Schneekloth, John S.

doi:10.1021/acs.biochem.7b01141

Cited by 75 publications

(83 citation statements)

References 26 publications

(59 reference statements)

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“…Despite the dynamics of RNA structure, both the A and U bulge regions in the miR-21 hairpin precursor have been observed by NMR (Chirayil, et al, 2014). Additional binding analyses using microscale thermophoresis (MST) (Moon, et al, 2018; Seidel, et al, 2013), indicated the binding affinity of 3 to a miR-21 hairpin containing both the A and U bulge (miR-21 Hairpin; Figure S2D) and a miR-21 hairpin containing only the A bulge (miR-21 A Bulge; Figure S2E) as 500 and 300 nM, respectively. Notably, no binding was observed to a miR-21 base pair control without the A and U bulges (miR-21 Base Pair; Figure S2F).…”

Section: Resultsmentioning

confidence: 99%

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs

Velagapudi

Costales

Vummidi

et al. 2018

Cell Chemical Biology

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Potential RNA drug targets for small molecules are found throughout the human transcriptome, yet small molecules known to elicit a pharmacological response by directly targeting RNA are limited to antibacterials. Herein, we describe AbsorbArray, a small molecule microarray-based approach that allows for unmodified compounds, including FDA-approved drugs, to be probed for binding to RNA motif libraries in a massively parallel format. Several drug classes bind RNA including kinase and topoisomerase inhibitors. The latter avidly bound the motif found in the Dicer site of oncogenic microRNA (miR)-21 and inhibited its processing both in vitro and in cells. The most potent compound de-repressed a downstream protein target and inhibited a miR-21-mediated invasive phenotype. The compound's activity was ablated upon overexpression of pre-miR-21. Target validation via chemical crosslinking and isolation by pull-down showed direct engagement of pre-miR-21 by the small molecule in cells, demonstrating that RNAs should indeed be considered druggable.

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Section: Resultsmentioning

confidence: 99%

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs

Velagapudi

Costales

Vummidi

et al. 2018

Cell Chemical Biology

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“…The RNA was annealed by heating to 95°C for 2 min and then cooled to room temperature. MST experiments were conducted in triplicate on a Monolith NT.115 system (NanoTemper Technologies) (45,46) and the normalized fluorescence (oFnorm) was determined from the MST traces for titration experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Then, the RNA was labeled with fluorescein-5-thiosemicarbazide as previously described (44). Aminoglycoside-to-fluorescent-RNA-binding measurements were made on a Monolith NT.115 system by NanoTemper Technologies (45,46); the results showed an increase in the measured response upon titration of the aminoglycosides, indicating the formation of an aminoglycoside-RNA complex. Figure 3B shows the dissociation constants (K d ) for the aminoglycoside-RNA complex formed, as measured by MST.…”

Section: Analysis Of the Relationship Between Integron Recombination mentioning

confidence: 99%

Integron-Derived Aminoglycoside-Sensing Riboswitches Control Aminoglycoside Acetyltransferase Resistance Gene Expression

Wang

Sun

et al. 2019

Antimicrob Agents Chemother

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Class 1 integrons accumulate antibiotic resistance genes by site-specific recombination at aatI-1 sites. Captured genes are transcribed from a promoter located within the integron; for class 1 integrons, the first gene to be transcribed and translated normally encodes an aminoglycoside antibiotic resistance protein (either an acetyltransferase [AAC] or adenyltransferase [AAD]). The leader RNA from the Pseudomonas fluorescens class 1 integron contains an aminoglycoside-sensing riboswitch RNA that controls the expression of the downstream aminoglycoside resistance gene. Here, we explore the relationship between integron-dependent DNA recombination and potential aminoglycoside-sensing riboswitch products of recombination derived from a series of aminoglycoside-resistant clinical strains. Sequence analysis of the clinical strains identified a series of sequence variants that were associated with class I integron-derived aminoglycoside-resistant (both aac and aad) recombinants. For the aac recombinants, representative sequences showed up to 6-fold aminoglycosidedependent regulation of reporter gene expression. Microscale thermophoresis (MST) confirmed RNA binding. Covariance analysis generated a secondary-structure model for the RNA that is an independent verification of previous models that were derived from mutagenesis and chemical probing data and that was similar to that of the P. fluorescens riboswitch RNA. The aminoglycosides were among the first antibiotics to be used clinically, and the data suggest that in an aminoglycoside-rich environment, functional riboswitch recombinants were selected during integron-mediated recombination to regulate aminoglycoside resistance. The incorporation of a functional aminoglycoside-sensing riboswitch by integron recombination confers a selective advantage for the expression of resistance genes of diverse origins.

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“…4C). [186][187][188][189][190][191][192] To quantify the intermolecular interactions, infrared-lasers are used to achieve precise microscale temperature gradients within the glass capillaries filled with the targeted solution, e.g. cell lysate, serum or bioliquids.…”

Section: Microscale Thermophoresis (Mst)mentioning

confidence: 99%

“…The Schneekloth laboratory has recently explored the utility of MST for probing HIV RRE-neomycin and HIV RRE-Rev peptide interactions. 189 They also examined competition in RRE RNA binding between neomycin and the Rev peptide via two different approaches. In all of these applications, MST effectively measured binding affinities and the results agreed with previously reported values determined via different biophysical techniques.…”

Section: Microscale Thermophoresis (Mst)mentioning

confidence: 99%

Unveiling the druggable RNA targets and small molecule therapeutics

Sztuba-Solińska

Chávez-Calvillo

Cline

2019

Bioorganic & Medicinal Chemistry

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Keywords:RNA structure and function RNA interactions Epitranscriptomics Small molecules RNA-based therapeutics RNA in disease Drug target A B S T R A C T The increasing appreciation for the crucial roles of RNAs in infectious and non-infectious human diseases makes them attractive therapeutic targets. Coding and non-coding RNAs frequently fold into complex conformations which, if effectively targeted, offer opportunities to therapeutically modulate numerous cellular processes, including those linked to undruggable protein targets. Despite the considerable skepticism as to whether RNAs can be targeted with small molecule therapeutics, overwhelming evidence suggests the challenges we are currently facing are not outside the realm of possibility. In this review, we highlight the most recent advances in molecular techniques that have sparked a revolution in understanding the RNA structure-to-function relationship. We bring attention to the application of these modern techniques to identify druggable RNA targets and to assess small molecule binding specificity. Finally, we discuss novel screening methodologies that support RNA drug discovery and present examples of therapeutically valuable RNA targets. RNA as a drug targetThe vast diversity of RNAs expanding beyond coding transcripts to several classes of ncRNAs that vary in length, biogenesis, polarity, and putative functions, increases the repertoire of druggable targets. 19,20 Here, specific RNA properties contribute to its attractive, yet challenging makeup as a target molecule. RNA can form complex three-dimensional structures through canonical Watson-Crick base pairing and complex tertiary interactions that are mediated by non-canonical bonds. Such structures can be as intricate and stable as those formed by proteins and can recognize small-molecule ligands, other nucleic acids, and/or proteins with high affinity and specificity. [21][22][23][24] At the same time, the highly dynamic conformation and repetitive character of its surface presents difficulties for drug design. 25,26 In addition, many putative small molecule-binding pockets in RNA are much more polar and solvent exposed than binding sites on proteins, complicating ligand design efforts. Compounding these issues, most target RNAs are expressed at low levels, with the exception of ribosomal (rRNA) and transfer (tRNA) RNAs, which constitute 80-90% and 10-15% of total cellular RNA, respectively. 27 Other abundant RNAs, such as messenger (mRNA), small nuclear (snRNA), and small nucleolar (snoRNA) are present at levels that are about 1-2 orders of magnitude lower than rRNA and tRNA. Certain small RNAs, such as micro (miRNA) and piwi (piRNAs) can be present at very high levels; however, this appears to be cell type dependent. One should keep in mind that if the RNA has catalytic activity or if it acts as a scaffold to regulate https://doi.

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Measuring RNA–Ligand Interactions with Microscale Thermophoresis

Cited by 75 publications

References 26 publications

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs

Integron-Derived Aminoglycoside-Sensing Riboswitches Control Aminoglycoside Acetyltransferase Resistance Gene Expression

Unveiling the druggable RNA targets and small molecule therapeutics

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