Rapid determination and quantitation of the accessibility to native RNAs by antisense oligodeoxynucleotides in murine cell extracts.
A major concern for antisense experiments is the prediction of effective oligonucleotide binding sites. We have developed a system to carry out oligodeoxyribonucleotide-RNA and ribozyme-RNA binding experiments in cell extracts to create a protein environment known to directly influence the structure of the mRNA. In these experiments the native, endogenous mRNA is probed using oligodeoxyribonucleotides (ODNs) to identify RNase H-accessible sites. The resulting RNase H-mediated cleavages in the cell extracts were quantified using RT-PCR with fluorescein and rhodaminetagged primers to generate fluorescent products that are analyzed and quantified on an automated DNA sequencer. As a model substrate for testing this system, we have targeted the murine DNA methyltransferase (MTase) mRNA. An ODN binding site in native MTase mRNA was identified that was cleaved by endogenous RNase H with an efficiency of 85% in the extracts. The ODN that was most effective in the cell extracts was also found to provide the best activity in vivo , resulting in a 75-85% reduction of the MTase mRNA. These data support the use of cell extracts and native transcripts to identify antisense and perhaps ribozyme target sites.
Scherr
M
M
Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3011, USA.
Rossi
J J
JJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002474:Cell-Free System; D015254:DNA Modification Methylases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics* / Q000378:metabolism*; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D014162:Transfection
D020319:Oligodeoxyribonucleotides, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012313:RNA; D015254:DNA Modification Methylases; D016914:Ribonuclease H