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|Title:||The Synthesis of Oligodeoxyribonucleotides With Rna Ligase|
|Author(s):||Hinton, Deborah Meetze|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||RNA ligase, isolated from bacteriophage T4-infected Escherichia coli catalyzes the ATP-dependent formation of a 3' (--->) 5'-phosphodiester bond between one oligonucleotide with a 3'-hydroxyl group (the acceptor molecule) and another with a 5'-phosphate (the donor molecule). The enzyme does not require the alignment of the donor and acceptor molecules onto a complementary template strand, and the reaction can yield an unique product. For these reasons, RNA ligase has proved useful for the synthesis of oligoribonucleotides of defined sequence. This work involves the extension of the RNA ligase reaction to DNA substrates and, in particular, the use of the single nucleotide addition reaction of RNA ligase with DNA acceptors.
A set of reaction conditions were determined for the use of RNA ligase with deoxyoligomer acceptors. These included high enzyme concentrations, long incubation times at low temperatures, the use of manganous rather than Mg(II) ion, and low ATP concentrations. Under these conditions, all common deoxyribonucleoside 3',5'-bisphosphates (pdAp, pdCp, pdGp, pdTp and pdUp) were donors and oligodeoxyribonucleotides with dA, dC, dG, dT, or dU 3' termini acted as acceptors. Product yields varied from 40 to 75%, and the products were characterized by paper chromatography and nearest neighbor analysis. Reaction conditions were improved by the use of a limiting ATP concentration coupled with an ATP regeneration system and the use of spermine and/or RNase A. Using these optimized reaction conditions, any common pdNp was added to the deoxyoligomer acceptor dA(pdA)(,4) with yields of (GREATERTHEQ) 85% in 1 to 10 days.
The use of RNA ligase for the practical synthesis of DNA was demonstrated by the synthesis of several oligodeoxyribonucleotides of defined sequence. Reactions, performed using substrate amounts of 1 to 200 nmol, resulted in single nucleotide addition yields of 13 to 95%, and demonstrated that RNA ligase can be used to add single, specific nucleotides to the 3' end of deoxyoligomers. In addition, the deoxyoligomer joining reaction of RNA ligase was used to ligate oligodeoxyribonucleotides which had been synthesized by a combination of chemical techniques and the RNA ligase single nucleotide addition reaction. The products were separated by paper and column chromatography and reverse phase high pressure liquid chromatography. Although reaction mixtures were incubated from 7 to 21 days, product characterizations demonstrated only slight degradation of product or substrates. Thus, we have shown that the reaction of RNA ligase with DNA substrates, coupled with other chemical and enzymatic techniques, is a practical method for the synthesis of oligodeoxyribonucleotides.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
|Date Available in IDEALS:||2014-12-14|