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A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an
O Matveeva1, B Felden, S Audlin
1Department of Human Genetics and Howard Hughes Medical Institute, 6160 Eccles Genetics Building, University of Utah, Salt Lake City, UT 84112, USA. olgam@howard.genetics.utah.edu
Nucleic Acids Research
|February 28, 1998
Summary
This study introduces a novel RNase H-based method to map accessible RNA sequences for DNA probe interactions. The technique accurately predicts effective antisense oligonucleotide targets by identifying cleavage sites on RNA molecules.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Identifying accessible RNA sequences is crucial for developing effective antisense therapies and molecular probes.
- Current methods may not accurately predict in vivo RNA accessibility and probe binding efficiency.
Purpose of the Study:
- To develop and validate a new technique for mapping RNA sequences accessible to complementary DNA probes.
- To assess the utility of this method for predicting efficient in vivo antisense targeting oligonucleotides.
Main Methods:
- Utilizing RNase H digestion of target RNA in the presence of random DNA fragments.
- Employing PCR amplification, DNase digestion, and denaturation of DNA prior to RNA binding.
- Detecting RNA cleavage sites via gel electrophoresis or primer extension.
Main Results:
- The method successfully mapped accessible sites on c-raf mRNA, correlating with known oligonucleotide activity.
- New susceptible sites in the 3'-untranslated region of c-raf mRNA were identified.
- Accessibility was evaluated across diverse RNA structures, including a group I intron, tRNA, tmRNA, and rRNA.
Conclusions:
- The described RNase H-based technique is a valuable tool for predicting in vivo antisense oligonucleotide efficacy.
- The method provides insights into RNA structure-accessibility relationships and identifies novel targeting sites.