Branched nucleic acid analogs were synthesized in order to investigate the substrate specificity of the yeast debranching enzyme (i.e. specific cleavage at the 2',5'-phosphodiester bond). In order to synthesize the branched nucleic acid analog dAA(G)TBDPSi (where the bracketed nucleoside is found at the 2'-position of A), the branch point nucleoside, N6-Bz-5'-O-MMTr-3 '-O-TBDPSi-adenosine-2'-O-[N,N-diisopropyl(cyanoethyl)] phosphoramidite was first synthesized. The branched trimer, dAA(G)TBDPSi was then assembled via solid phase synthesis using an automated DNA synthesizer. Another nucleic acid analog, dAA(G)PO42- was also synthesized as was its corresponding expected debranched product, dAA(OH)PO 42-. The known yDBR enzyme substrate, dAA(G)G was synthesized as well, along with its debranched product dAA(OH)G so that it could be used as a positive control in yDBR enzyme studies. MALDI-TOF MS and PAGE were utilized to characterize the synthesized oligonucleotides. / dAA(G)PO42- and dAA(G)G were radiolabelled and subjected to the yDBR enzyme and the autoradiograph was analyzed. dAA(G)PO 42- was in fact cleaved by the yDBR enzyme, although not nearly as efficiently as the fully branched dAA(G)G tetramer.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.33421 |
| Date | January 2000 |
| Creators | Liscio, Anita. |
| Contributors | Damha, Masad J. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001779367, proquestno: MQ70728, Theses scanned by UMI/ProQuest. |
Page generated in 0.0016 seconds