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In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase
1Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and VA Connecticut Healthcare System, New Haven, CT 06520, USA.
Nucleic Acids Research
|November 1, 2000
Summary
Researchers identified a novel RNA molecule that binds human thymidylate synthase (TS) with high affinity. This RNA enhances gene expression and protein production in cancer cells, offering potential therapeutic targets.
Area of Science:
- Molecular Biology
- RNA Therapeutics
- Cancer Research
Background:
- Thymidylate synthase (TS) translation is repressed by TS binding to its own mRNA.
- Identifying specific RNA sequences that bind TS is crucial for understanding this regulation.
Purpose of the Study:
- To discover novel RNA sequences with high affinity for human TS protein.
- To investigate the functional impact of these RNA sequences in cancer cells.
Main Methods:
- In vitro synthesis of a degenerate RNA pool and subsequent in vitro selection.
- Secondary structure analysis, deletion/modification assays, and in vivo transfection experiments.
- Immunoprecipitation coupled with RT-PCR to confirm direct RNA-protein interaction.
Main Results:
- A single RNA molecule was selected with 20-fold higher affinity for TS than wild-type sequences.
- A UGU loop element was critical for binding; its modification reduced affinity by 1000-fold.
- The selected RNA increased reporter gene expression and TS protein levels in human colon cancer cells.
Conclusions:
- A novel, high-affinity RNA ligand for human TS was identified.
- This RNA sequence specifically interacts with TS and modulates its expression in cancer cells.
- The findings suggest potential applications for RNA-based therapeutics targeting TS.