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|Title:||DNA as a Catalyst and as a Sensor: Investigating the Structure of the Ty1 Retrotransposition Intermediate and the Utility of Dna-Based Biosensors|
|Author(s):||Pratico, Elizabeth D.|
|Doctoral Committee Chair(s):||Silverman, Scott K.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The catalytic and binding properties of functional nucleic acids have been exploited to investigate key biochemical questions and to detect small molecules for diagnostic applications. Catalytic DNA (deoxyribozymes) can catalyze many different chemical reactions such as RNA ligation, RNA cleavage, DNA phosphorylation, DNA ligation, DNA adenylation, nucleopeptide formation, Diels-Alder reaction, and porphyrin metalation. RNA ligation is catalyzed by using either an internal 2'-hydroxyl, a terminal 3'-hydroxyl or a terminal 2'-hydroxyl to attack a 5'-triphosphate, synthesizing a variety of RNA linkages: 2',5'-branch, 3'-5'-linear, or 2'-5'-linear. RNA-branching deoxyribozymes can be used to study the role of specific biochemical intermediates, such as the putative 2',5'-branched RNA intermediate in Ty1 retrotransposition. Aptamers are three-dimensional structures of single-stranded DNA that can bind small molecules with both high affinity and selectivity. Aptamers are isolated by in vitro selection from random libraries of single-stranded sequences by their ability to recognize a particular target. The aptamer can then be developed into a sensor, which can be used as a diagnostic tool for the detection of a specific target.
2',5'-Branched RNA was recently proposed as a key Ty1 retrotransposition intermediate, for which cleavage by lariat debranching enzyme (Dbr1p) enables reverse transcription to continue synthesizing the complete Ty1 cDNA. Since a substantial amount of cDNA (18%) is produced by dbr1 cells lacking Dbr1p, the branched RNA intermediate would require that Ty1 reverse transcriptase (RT) can read through the proposed branch site. Using a two-step strategy involving the deoxyribozymes 6CE8 and 10-23, we have synthesized the proposed Ty1 2',5'-branched RNA for a direct test of Ty1 RT's read-through ability. Using an in vitro assay that incorporates all components known to be required for Ty1 cDNA synthesis (including the TyA chaperone protein), Ty1 RT can elongate up to the branch site, and strand transfer from the 2'-arm to the 3'-arm of the branch is observed. However, when elongating from either the 2'-arm or the 3'-arm, Ty1 RT reads through the branch site with only ∼0.3% efficiency, which is ≥60-fold lower than would be necessary to explain the Ty1 cDNA synthesized in dbr1 cells in vivo. Our finding that Ty1 RT cannot read through the proposed Ty1 branch site is inconsistent with the originally proposed hypothesis that branched RNA is a key intermediate for the first steps of cDNA synthesis found in Ty1 retrotransposition. Our investigations and others suggest that Dbr1p acts other than as a 2',5'-phosphodiesterase during Ty1 retrotransposition.
In vitro selection has been utilized to develop aptamers and aptazymes as components of small-molecule sensors. Aptamers have been selected for a range of targets from proteins to small-molecule ligands. Aptazymes have an aptamer domain that can allosterically control the catalysis of an attached nucleic acid enzyme. The targets for our sensors are mycotoxins, which are secondary metabolites of fungi. Mycotoxins have been found in crops during harvesting and storage. Consumption of mycotoxin-contaminated crops can lead to toxic effects in humans and animals, making it necessary to survey crops for these toxins. We have pursued two strategies to identify mycotoxin-sensing DNA aptamers and aptazymes, which will later be developed into on-site reporter systems. The first strategy was to implement direct selections for mycotoxin-dependent RNA-cleaving aptazymes. The second strategy was to conduct a selection for a mycotoxin aptamer that can later be appended to an RNA-cleaving deoxyribozyme through a communication module, which can be rationally designed or identified by in vitro selection. Aptamers with high affinities (K d <2 microM) have been identified for estradiol (E2) and zearalenone (ZEN). Conversion of the estradiol and zearalenone aptamers into sensors will utilize two different approaches: structure-switching signaling aptamers (switchamers) and aptazymes.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
|Date Available in IDEALS:||2014-12-17|