Biology subjects

Balaratnam, S.

Publications and source records attributed to Balaratnam, S..

4 recordsLinked to original sources

bioRxiv2025

Discovery of small molecule probes targeting the 5' stem-loop in the yeast U4/U6 snRNA

The spliceosome is a large ribonucleoprotein complex that regulates pre-mRNA splicing and has been an intriguing target for drug discovery. Essential to the assembly of the spliceosome are the small nuclear RNAs (snRNAs), which form RNA-RNA and RNA-protein interactions in the intact spliceosome and during its assembly. Here, we study the yeast U4/U6 snRNA assembly and report the rapid discovery of small molecule K-turn ligands via parallel small molecule microarray (SMM) screening of multiple related RNA constructs. For hit validation, biophysical analyses were conducted to confirm the binding and effects on thermodynamic stability and of RNA structure. One analog of the hit molecule (22) exhibited improved affinity towards the yeast U4/U6 snRNA (KD = 3.9 {+/-} 2.2 {micro}M). The specific interaction between 22 and the K-turn region was studied experimentally using deltaSHAPE and in silico with MD simulations. Moreover, this molecule was found to inhibit the binding of the U4 to Snu13 in biochemical assays (IC50 = 3.2 {+/-} 0.4 {micro}M). This work reports new ligands for the U4 snRNA and reveals that a multiplexed, structure-based approach can be used to identify small molecules that bind to specific regions of complex RNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/681657v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c156f4org.highwire.dtl.DTLVardef@133e90eorg.highwire.dtl.DTLVardef@109b793org.highwire.dtl.DTLVardef@f3301f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Bioorthogonal cyclopropenones for investigating RNA structure

RNA sequences encode secondary and tertiary structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but the complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA and trap native conformations in cellular environments. Existing tools primarily rely on electrophiles that are constitutively "on" or triggered by UV light, often resulting in high background reactivity. We developed an alternative, chemically triggered approach to crosslink RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes--electrophiles that can trap neighboring nucleophiles to forge covalent crosslinks. As proof-of-concept, we synthesized a panel of CpOs and appended them to thiazole orange (TO-1). The TO-1 conjugates bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, confirmed by fluorescence turn-on. After phosphine administration, covalent crosslinks were formed between the CpO probes and RNA. The degree of crosslinking was both time and dose-dependent. We further applied the chemically triggered tools to model RNAs in biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.

biochemistry↗

bioRxiv2023

Competitive Microarray Screening Reveals Functional Ligands for the DHX15 RNA G-quadruplex

RNAs are increasingly considered valuable therapeutic targets, and in turn the development of methods to identify and validate both RNA targets and RNA-binding compounds is more important than ever. In this study, we utilized a bioinformatic approach to identify a hairpin-containing RNA G-quadruplex (rG4) in the 5' UTR of DHX15 mRNA. By using a competitive small molecule microarray (SMM) approach, we identified a compound that specifically binds to the DHX15 rG4 with a KD of 12.6 {+/-} 1 {micro}M. This rG4 directly impacts translation of a DHX15 reporter mRNA in vitro, and binding of our compound (F1) to the structure inhibits translation up to 57% with an IC50 of 22.9 {+/-} 3.8 {micro}M. The DHX15 protein is an "undruggable" helicase associated with several types of cancer progression, and our data represent the first published effort to target the rG4 in DHX15 mRNA to inhibit its translation. Overall, our work is informative for the development of novel small molecule cancer therapeutics for RNA targets starting from target identification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/550542v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@19eb40borg.highwire.dtl.DTLVardef@3e6a17org.highwire.dtl.DTLVardef@1a71f9eorg.highwire.dtl.DTLVardef@10ce4df_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Investigating the NRAS 5' UTR as a Target for Small Molecules

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. Constitutively activated NRAS induces the MAPK and AKT signaling pathways and leads to uncontrolled proliferation and cell growth, making it an attractive target for small molecule inhibition. Like all RAS-family proteins, it has proven difficult to identify small molecules that directly inhibit the protein. An alternative approach would involve targeting the NRAS mRNA. The 5' untranslated region (5' UTR) of the NRAS mRNA is reported to contain a G-quadruplex (G4) that regulates translation of NRAS mRNA. Stabilizing the G4 structure with small molecules could reduce NRAS protein expression in cancer cells by impacting translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5' UTR of the NRAS mRNA. We used a small molecule microarray (SMM) screen to identify molecules that selectively bind to the NRAS-G4. Biophysical studies demonstrated that compound 18 binds reversibly to the NRAS-G4 structure with submicromolar affinity. A Luciferase based reporter assay indicated that 18 inhibits the translation of NRAS via stabilizing the NRAS-G4 in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends (RACE), RT-PCR analysis on 14 different NRAS-expressing cell lines, coupled with analysis of publicly available CAGE seq experiments, revealed that predominant NRAS transcript does not possess the G4 structure. Further analysis of published rG4 and G4 sequencing data indicated the presence of G4 structure in the promoter region of NRAS gene (DNA) but not in the mRNA. Thus, although many NRAS transcripts lack a G4 in many cell lines the broader concept of targeting folded regions within 5 UTRs to control translation remains a highly attractive strategy and this work represents an intriguing example of transcript heterogeneity impacting targetability.

biochemistry↗