Related Experiment Video
Updated: May 2, 2026
An Assay for Quantifying Protein-RNA Binding in Bacteria
Published on: June 12, 2019
Assessing specific oligonucleotides and small molecule antibiotics for the ability to inhibit the CRD-BP-CD44 RNA
Dustin T King1, Mark Barnes1, Dana Thomsen1
1Chemistry Program, University of Northern British Columbia, Prince George, British Columbia, Canada.
Abstract:
Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3'UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862-3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862-3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.
Insights
Coding Region Determinant-Binding Protein (CRD-BP) regulates mRNA stability and is overexpressed in cancers. This study identifies specific antisense oligonucleotides and aminoglycoside antibiotics that inhibit the CRD-BP-CD44 RNA interaction, offering potential therapeutic strategies.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- Coding Region Determinant-Binding Protein (CRD-BP) is implicated in mRNA stability and localization.
- CRD-BP is minimally expressed in normal adult tissues but overexpressed in aggressive human cancers and neonatal tissues.
- CRD-BP's oncogenic role is linked to its physical association with target mRNAs, notably CD44.
Purpose of the Study:
- To further characterize the interaction between CRD-BP and CD44 mRNA.
- To assess the efficacy of antisense oligonucleotides and small molecule antibiotics in inhibiting this interaction.
- To explore potential therapeutic strategies targeting the CRD-BP-CD44 RNA interaction.
Main Methods:
- In vitro binding assays to determine CRD-BP affinity for CD44 RNA.
- Screening of antisense oligonucleotides for their ability to compete with CRD-BP binding.
- Evaluation of aminoglycoside antibiotics for inhibition of the CRD-BP-CD44 RNA interaction.
- Cell-based assays to assess the impact of inhibitors on CD44 mRNA levels.
Main Results:
- CRD-BP exhibits high-affinity binding to CD44 RNA (Kd of 645 nM).
- Three antisense oligonucleotides (DD4, DD7, DD10) effectively competed with CRD-BP binding and reduced CD44 mRNA levels in cells.
- Aminoglycoside antibiotics (neomycin, paramomycin, kanamycin, streptomycin) inhibited the interaction in vitro, though cell-based assessment was challenging due to non-specific binding.
Conclusions:
- Specific antisense oligonucleotides can effectively inhibit the CRD-BP-CD44 RNA interaction.
- Aminoglycoside antibiotics show potential as inhibitors, but further investigation is needed for cell-specific effects.
- These findings provide a basis for developing novel small molecule and nucleic acid-based inhibitors targeting protein-RNA interactions in cancer.
More Related Videos
07:55An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA
Published on: February 17, 2023
06:43A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents
Published on: August 31, 2013
