A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures

Abstract

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.

Comparative Study of DNA Enzymes and Ribozymes against the Same Full-length Messenger RNA of the Vanilloid Receptor Subtype I

“…An alternative approach was the use of random or semirandom ODN libraries and RNase H followed by primer extension (31, 32). In a nonrandom variation of this strategy target-specific oligonucleotides were generated by digestion of the template DNA (33). All of these methods are labor intensive and expensive due to the primer extension analysis and do not reveal further information to the rather simple messenger walk screening presented in this study.…”

Complete, gene-specific siRNA libraries: Production and expression in mammalian cells

“…Under these conditions DNase I displays little sequence specificity, cleaving all regions of the DNA (except the terminal nucleotides) at an equal rate (Anderson 1981). Since DNase I generates fragments with a wide size distribution, reaction time and temperature were varied to determine optimal conditions to maximize the proportion of DNA in the desired size range (Anderson 1981;Matveeva et al 1997). Aliquots were collected at various time points and quenched with an equal volume of loading buffer (95% formamide, 10 mM EDTA, 0.1% SDS) and DNA fragments corresponding to 20-30 bp were isolated by native 15% polyacrylamide gel.…”

Oligodeoxyribozymes That Cleave β-Catenin Messenger RNA Inhibit Growth of Colon Cancer Cells via Reduction of β-Catenin Response Transcription

“…Antisense oligonucleotides should be designed by avoiding the underlying secondary structure of target RNA, which hampers the accessibility of antisense oligonucleotides. Prediction of the location of unpaired loops does not guarantee effective hybridization sites due to unpredictable steric and topological constraints of long stretches of RNA (20,21). To overcome this problem, we previously designed a method to identify accessible cleavage sites in the long target RNA using a pool of oligodeoxyribozymes which contains randomized sequences for RNA binding (19).…”