Creation of RNA molecules that recognize the oxidative lesion 7,8-dihydro-8-hydroxy-2'-deoxyguanosine (8-oxodG) in DNA.
We used in vitro evolution to obtain RNA molecules that specifically recognize and bind with high affinity to the oxidative lesion 7, 8-dihydro-8-hydroxy-2'-deoxyguanosine (8-oxodG) in DNA. A pool of approximately 10(15) RNA molecules containing a random insert of 45 nucleotides in length was subject to 10 successive rounds of chromatographic enrichment using an 8-oxodG affinity matrix, reverse transcription, PCR amplification, and RNA synthesis. Selected RNA molecules bind to 8-oxodG located at the 3' terminus (Kd </= 270 nM) or in the center (Kd </= 2.8 microM) of a 19-nt strand of DNA, with no detectable affinity for the corresponding dG-containing DNA sequences. These 8-oxodG-binding RNAs will be used to monitor levels of 8-oxodG in DNA from biological sources and should provide a unique method for evaluating oxygen-mediated DNA damage. This approach should be applicable for the creation of RNA molecules that can bind to and identify the different modifications of DNA produced by a variety of environmental agents.
Rink
S M
SM
The Joseph Gottstein Memorial Cancer Research Laboratory, Departments of Pathology and Biochemistry, University of Washington School of Medicine, Seattle, WA 98195-7705, USA.
Shen
J C
JC
Loeb
L A
LA
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019020:Directed Molecular Evolution; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D012313:RNA / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D012313:RNA; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine