A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures.
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.
Matveeva
O
O
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
Felden
B
B
Audlin
S
S
Gesteland
R F
RF
Atkins
J F
JF
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000096:biosynthesis*; D004275:DNA, Ribosomal / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D012313:RNA / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012349:RNA, Transfer, Asp / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D051381:Rats; D016914:Ribonuclease H / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D013768:Tetrahymena / Q000235:genetics
D018076:DNA, Complementary; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; D012349:RNA, Transfer, Asp; C096222:tmRNA; D012313:RNA; D019908:Proto-Oncogene Proteins c-raf; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H