RNA aptamers targeting the carboxyl terminus of KRAS oncoprotein generated by an improved SELEX with isothermal RNA amplification.
Mutations in the KRAS gene occur frequently in various human tumors and are known to lead to malignant transformation. We isolated RNA aptamers targeting activated mutant KRAS proteins using an improved SELEX method by isothermal RNA amplification. RNA aptamers were selected against mutant KRAS (G12V) proteins, as well as a biotinylated 15-amino-acid peptide from the carboxyl terminal of KRAS that contains a farnesylation site. All the selected RNA aptamers bound to the basic carboxy-terminal region of KRAS protein and the highest K(D) value was 2.3 microM. By an in vitro scintillation proximity assay, we demonstrated that KRAS aptamers inhibited farnesylation moderately. From these aptamers, we determined a consensus sequence (U)CCAAGCAC(AC) that, when concatamerized, exhibited higher binding affinity to the carboxy-terminal region of KRAS protein. Further improvement of binding affinity between aptamers and KRAS protein might provide a new therapeutic approach for activated mutant KRAS proteins.
Tanaka
Yoichiro
Y
Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama 362-0806, Japan., Department of Basic Medical Science, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Akagi
Kiwamu
K
Nakamura
Yoshikazu
Y
Kozu
Tomoko
T
D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry*; D001483:Base Sequence; D016384:Consensus Sequence; D006801:Humans; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry*; D052156:SELEX Aptamer Technique / Q000379:methods*; D013696:Temperature; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics
D052157:Aptamers, Nucleotide; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D012313:RNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins