scholarly journals Revealing the high propensity of RNAs to non-specifically bind drug-like small molecules

2020 ◽  
Author(s):  
Megan L. Kelly ◽  
Chia-Chieh Chu ◽  
Honglue Shi ◽  
Laura R. Ganser ◽  
Hal P. Bogerd ◽  
...  
Keyword(s):  
Small Molecules ◽  
Specific Binding ◽  
Cellular Activity ◽  
Response Element ◽  
Stem Loop ◽  
Rev Response Element ◽  
Nmr Structures ◽  
Important Challenge ◽  
Rna Binders

ABSTRACTIdentifying small molecules that selectively bind a single RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to non-specific binding of aminoglycosides and intercalators to a variety of RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities, however the ability of such compounds to discriminate against RNA stem-loops commonly found in the transcriptome has not been thoroughly assessed in all cases. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activity of three distinct RNAs, to non-specifically bind two HIV-1 stem-loop RNAs: the transactivation response element (TAR) and stem IIB in the rev response element (RREIIB). All three compounds bound to TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting substantial off-target interactions consistent with non-specific cellular activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, much like aminoglycosides, and in contrast to ligands that specifically bind riboswitches. Our results support extending the group of non-selective RNA-binders beyond aminoglycosides and intercalators to encompass drug-like compounds with capacity for non-specific hydrogen-bonding and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

scholarly journals Probing RNA conformational equilibria within the functional cellular context

2019 ◽  
Author(s):  
Laura R. Ganser ◽  
Chia-Chieh Chu ◽  
Hal P. Bogerd ◽  
Megan L. Kelly ◽  
Bryan R. Cullen ◽  
...  
Keyword(s):  
Biological Activities ◽  
Cellular Activity ◽  
Response Element ◽  
Indirect Measure ◽  
Rev Response Element ◽  
Cellular Environment ◽  
Cellular Context ◽  
Regulatory Rnas ◽  
Substitution Mutations

SummaryMany regulatory RNAs undergo changes in their structure from the dominant ground-state (GS) toward short-lived low-abundance ‘excited-states’ (ES) that reorganize local elements of secondary structure. ESs are increasingly observedin vitroand implicated in the folding and biological activities of regulatory RNAs and as targets for developing therapeutics. However, whether these ESs also form with comparable abundance within the complex cellular environment remains unknown. Here, we developed an approach for assessing the relative stability and abundance of RNA ESs within the functional cellular context. The approach uses point substitution mutations to increase the population of an inactive ES relative to the active GS. The cellular activity of such ES-stabilizing mutants then provides an indirect measure of any residual population of the active GS within the functional cellular context. Compensatory rescue mutations that restore the GS are used to control for changes in cellular activity arising due to changes in sequence. The approach is applied to probe ESs in two highly conserved and functionally important regulatory RNAs from HIV-1: the transactivation response element (TAR) and the Rev response element (RRE). For both RNAs, ES-stabilizing mutations inhibited cellular activity to a degree that correlates with the extent to which they stabilize the ES relative to the GSin vitro. These results indicate that the non-native ESs of TAR and RRE likely form in cells with abundances comparable to those measuredin vitroand their targeted stabilization provides a new avenue for developing anti-HIV therapeutics.

scholarly journals Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1517
Author(s):  
Rebecca S. Brown ◽  
Lisa Kim ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Rna Structure ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Specific Binding ◽  
Genomic Rna ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Labeled Rna

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

scholarly journals Conserved Stem II of the Box C/D Motif Is Essential for Nucleolar Localization and Is Required, Along with the 15.5K Protein, for the Hierarchical Assembly of the Box C/D snoRNP

2002 ◽  
Vol 22 (23) ◽  
pp. 8342-8352 ◽  
Author(s):  
Nicholas J. Watkins ◽  
Achim Dickmanns ◽  
Reinhard Lührmann
Keyword(s):  
Specific Binding ◽  
Nucleolar Localization ◽  
Homologous Proteins ◽  
Stem Loop ◽  
Hierarchical Assembly ◽  
Conserved Sequence ◽  
Associated Proteins ◽  
The Individual ◽  
Stem Structure

ABSTRACT The 5′ stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem II of the box C/D motif, in contrast to the U4 5′ stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem II determines the specific assembly of the box C/D snoRNP.

HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region

Cell ◽  
1990 ◽  
Vol 60 (4) ◽  
pp. 685-693 ◽  
Author(s):  
Shaun Heaphy ◽  
Colin Dingwall ◽  
Ingemar Ernberg ◽  
Michaet J. Gait ◽  
Sheila M. Green ◽  
...  
Keyword(s):  
Loop Structure ◽  
Response Element ◽  
Stem Loop ◽  
Rev Response Element ◽  
Stem Loop Structure ◽  
Hiv 1 ◽  
Rev Protein

scholarly journals Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display

2004 ◽  
Vol 384 (3) ◽  
pp. 585-590 ◽  
Author(s):  
Aiguo XU ◽  
David Li-En JAO ◽  
Kuang Yu CHEN
Keyword(s):  
Differential Display ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Deoxyhypusine Synthase ◽  
Mobility Shift ◽  
Arbitrary Primers ◽  
Stem Loop ◽  
Deoxyhypusine Hydroxylase ◽  
Rev Response Element

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555–2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35a, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.

scholarly journals Self–assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed

1999 ◽  
Vol 354 (1383) ◽  
pp. 537-550 ◽  
Author(s):  
P. J. G. Butler
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Self Assembly ◽  
Specific Binding ◽  
Macromolecular Structure ◽  
Stem Loop ◽  
Protein Subunits ◽  
Internal Site

The tobacco mosaic virus (TMV) particle was the first macromolecular structure to be shown to self–assemble in vitro , allowing detailed studies of the mechanism. Nucleation of TMV self–assembly is by the binding of a specific stem–loop of the single–stranded viral RNA into the central hole of a two–ring sub–assembly of the coat protein, known as the ‘disk’. Binding of the loop onto its specific binding site, between the two rings of the disk, leads to melting of the stem so more RNA is available to bind. The interaction of the RNA with the protein subunits in the disk cause this to dislocate into a proto–helix, rearranging the protein subunits in such a way that the axial gap between the rings at inner radii closes, entrapping the RNA. Assembly starts at an internal site on TMV RNA, about 1 kb from its 3′–terminus, and the elongation in the two directions is different. Elongation of the nucleated rods towards the 5′–terminus occurs on a ‘travelling loop’ of the RNA and, predominantly, still uses the disk sub–assembly of protein subunits, consequently incorporating approximately 100 further nucleotides as each disk is added, while elongation towards the 3′–terminus uses smaller protein aggregates and does not show this ’quantized‘ incorporation.

scholarly journals ID: 1083 Targeting the production of oncogenic miRNAs using synthetic small molecules

2017 ◽  
Vol 4 (S) ◽  
pp. 170
Author(s):  
Anh Thi-Phuong Tran ◽  
Duc Huy Vo ◽  
Audrey Di Giorgio ◽  
Maria Duca
Keyword(s):  
Small Molecules ◽  
Transcriptional Regulators ◽  
Mature Mirnas ◽  
Aberrant Expression ◽  
Stem Loop ◽  
Small Molecule Drugs ◽  
Non Coding Rnas ◽  
Rna Ligands ◽  
Oncogenic Mirnas

MicroRNAs (miRNAs or miRs) are a class of evolutionary conserved small non-coding RNAs that act as post-transcriptional regulators of gene expression. A wide number of studies has shown that the aberrant expression of miRNAs could be responsible for initiation and development of human cancers. Most of these deregulated miRNAs are overexpressed, thus being oncogenic. For these reasons, the inhibition of oncogenic miRNAs function or production would be a very promising approach for the development of new anticancer therapies [1]. The purpose of this work is the discovery of small-molecule drugs targeting the precursors of specific oncogenic miRNAs thus modulating their production. We have focused our attention on miRNA-372 and miRNA- 373 that are implicated in various cancers. For example, these two oncogenics (pre-miRNAs 372 and pre-miRNAs 373): two stem-loop structured RNAs that lead to mature miRNAs after cleavage by the enzyme Dicer. In the aim of inhibiting this biogenesis step and based on our previous works [2], we synthesized a novel series of RNA ligands composed of two different domains: (i) 2-deoxystreptamine (2-DOS) known as RNA-interacting group due to its role as a central scaffold of any known aminoglycosides [3] and (ii) aromatic or heteroaromatic groups that should improve affinity and selectivity of these ligands for the targeted RNAs. These two domains have been conjugated through carbamate, triazole and ether bonds in order to develop a simple and straightforward synthetic methodology and obtain a large number of diversified compounds.   We obtained a library of 16 compounds which will be evaluated in vitro in order to identify the strongest RNA ligands that could be able to inhibit the production of the targeted oncogenic miRNAs and eventually lead to the inhibition of cancer cells proliferation.

scholarly journals Identification of Tau Stem Loop RNA Stabilizers

2007 ◽  
Vol 12 (6) ◽  
pp. 789-799 ◽  
Author(s):  
Christine P. Donahue ◽  
Jake Ni ◽  
Eriks Rozners ◽  
Marcie A. Glicksman ◽  
Michael S. Wolfe
Keyword(s):  
Small Molecules ◽  
Binding Assay ◽  
Loop Structure ◽  
Stem Loop ◽  
Binding Domains ◽  
Stem Loop Structure ◽  
Exon 10 ◽  
Tau Expression

Alternative splicing of tau exon 10 produces tau isoforms with either 3 (3R) or 4 (4R) repeated microtubule-binding domains. Increased ratios of 4R to 3R tau expression, above the physiological 1:1, leads to neurofibrillary tangles and causes neurodegenerative disease. An RNA stem loop structure plays a significant role in determining the ratio, with decreasing stability correlating with an increase in 4R tau mRNA expression. Recent studies have shown that aminoglycosides are able to bind and stabilize the tau stem loop in vitro, suggesting that other druglike small molecules could be identified and that such molecules might lead to decreased exon 10 splicing in vivo. The authors have developed a fluorescent high-throughput fluorescent binding assay and screened a library of ∼110,000 compounds to identify candidate drugs that will bind the tau stem loop in vitro. In addition, they have developed a fluorescent-based RNA probe to assay the stabilizing effects of candiate drugs on the tau stem loop RNA. These assays should be applicable to the general problem of identifying small molecules that interact with mRNA secondary structures. ( Journal of Biomolecular Screening 2007:789-799)

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