CHEMICAL SYNTHETIC OLIGODEOXYRIBONUCLEOTIDES: PREPARATION, PURIFICATION, AND USE IN THE CHARACTERIZATION OF THE ACTIVITY OF ESCHERICHIA COLI RIBONUCLEASE H
Abstract
Several oligonucleotides were prepared by liquid phase phosphotriester and solid phase phosphoramidite methodologies. Solubility problems were encountered with the liquid phase chemistry which were solved by the inclusion of alkyl alcohols in the currently employed solvents for the chemically protected oligonucleotides. Syntheses carried out on glass supports with ribonucleoside linkers revealed that the use of supports with ribonucleosides linked to the support via the 5'-hydroxyl resulted in higher yields and cleaner reactions than supports with ribonucleosides linked via 2'(3')-hydroxyls. It was also determined that the currently used technique of measuring reaction yields by the release of the dimethoxytrityl group is unreliable, with little correlation evident between the actual composition of the final reaction mixtures and the results expected on the basis of the measurements. A new HPLC column system for rapid separations of synthetic oligonucleotides was developed. The column consists of polyethylene imine crosslinked over microparticulate silica. The column was found to be useful for analyzing mixtures from oligonucleotide syntheses, as well as for rapid purification of large quantities of oligonucleotides. Preparative separations of synthetic oligonucleotides were accomplished with near one hundred percent purity and generally high recoveries. The synthetic oligodeoxyribonucleotides were used to characterize the activity of ribonuclease H from Escherichia coli. Particular attention was given to the significance of the size and quantity of the DNA molecules complementary to the RNA substrate. The major cleavages occurred after the fifth or sixth base in the RNA from the 5' side of the RNA in the hybrids. The highest degree of specificity of cleavage was obtained in the presence of DNA molecules of six and seven bases at DNA:RNA ratios of 10:1. This specificity was independent of enzyme quantity. The activity of the enzyme was inhibited by both large quantities of single-stranded DNA molecules of eight bases and by DNA-RNA duplexes of six base pairs and greater. Ribonuclease H activity was found to be inhibited by the presence of secondary structures in the RNA substrate, and this characteristic was used to probe for the presence of RNA-RNA duplexes in six regions of E. coli 5S RNA.
Degree
Ph.D.
Subject Area
Biochemistry
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