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Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
Published on: January 16, 2020
Identification of RNA aptamer specific to mutant KRAS protein
Sujin Jeong1, Seung Ryul Han, Young Ju Lee
1Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Yongin, Republic of Korea.
Abstract:
Mutations in the KRAS gene are required for early occurrence and maintenance of tumorigenesis and are the most frequently found in many types of human malignant diseases. Therefore, approaches targeting RAS function have been proposed for cancer therapy. However, no selective and specific inhibitors of KRAS have yet been developed as anticancer agents. In this study, by employing counter-systematic evolution of ligands by exponential enrichment technique, we identified and characterized an RNA aptamer that specifically bound to mutant KRAS protein with a point mutation in codon 12 of the KRAS gene. Real-time polymerase chain reaction analysis, surface plasmon resonance measurements, and competitive precipitation experiments showed that the selected aptamer contained activities of specific and high-affinity binding to the mutant KRAS (K(D) approximately 4.04 nM) but much less binding to the wild type (K(D) approximately 227 nM). This RNA aptamer could be useful as a ligand for specific therapeutics and diagnostics against mutant KRAS-mediated cancers.
Insights
Researchers developed a novel RNA aptamer targeting the KRAS gene mutation. This specific aptamer shows high affinity for mutant KRAS, offering potential for targeted cancer therapeutics and diagnostics.
Area of Science:
- Molecular Biology
- Oncology
- Biotechnology
Background:
- KRAS gene mutations are crucial drivers in many human cancers, making RAS pathway inhibition a key therapeutic strategy.
- Despite the importance of KRAS, selective inhibitors for anticancer drug development remain elusive.
Purpose of the Study:
- To identify and characterize a novel RNA aptamer with specific binding affinity for mutant KRAS proteins.
- To evaluate the potential of this aptamer as a therapeutic or diagnostic agent for KRAS-mediated cancers.
Main Methods:
- Counter-systematic evolution of ligands by exponential enrichment (SELEX) was employed to discover the aptamer.
- Binding specificity and affinity were assessed using real-time PCR, surface plasmon resonance (SPR), and competitive precipitation assays.
Main Results:
- An RNA aptamer was identified that exhibits high-affinity binding (K(D) ≈ 4.04 nM) specifically to mutant KRAS (codon 12).
- The aptamer demonstrated significantly lower binding affinity to wild-type KRAS (K(D) ≈ 227 nM), confirming its specificity.
Conclusions:
- The developed RNA aptamer is a highly specific ligand for mutant KRAS.
- This aptamer holds promise for the development of targeted cancer therapeutics and diagnostic tools against KRAS-driven malignancies.
