Nucleic Acids Research, Vol 25, Issue 24 5010-5016, Copyright © 1997 by Oxford University Press
O Matveeva, B Felden, S Audlin, RF Gesteland and JF Atkins
A technique is described to identify the rare sequences within an RNA
molecule that are available for efficient interaction with complementary
DNA probes: the target RNA is digested by RNase H in the presence of a
random pool of complementary DNA fragments generated from the same DNA
preparation that was used for target RNA synthesis. The DNA region was
amplified by PCR, partially digested with DNase and denatured prior to RNA
binding. In the presence of single-stranded DNA fragments the RNA was
digested with RNase H such that, on average, each molecule was cut once.
Cleavage sites were detected by gel electrophoresis either directly with
end-labeled RNA or by primer extension. The pattern of accessible sites on
c- raf mRNA was determined and compared with the known profile of activity
of oligonucleotides found in cells, showing the merit of the method for
predicting oligonucleotides which are efficient for in vivo antisense
targeting. New susceptible sites in the 3'-untranslated region of c- raf
mRNA were identified. Also, four RNAs were probed to ascertain to what
extent structure predicts accessibility: the P4-P6 domain of the
Tetrahymena group I intron, yeast tRNAAsp, Escherichia coli tmRNA and a
part of rat 18S rRNA.
ARTICLES
A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures
Department 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
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