Date of Award

1-1-2013

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (v, 42 pages) : illustrations (some color)

Dissertation/Thesis Chair

Hua Shi

Committee Members

Richard Zitomer, Albert Millis

Keywords

Aptamer, RNA

Subject Categories

Molecular Biology

Abstract

Aptamers are oligonucleotide molecules (DNA or RNA) that are selected against specific target proteins, nucleic acids or small molecules by using the well established technique of SELEX (Systemic Evolution of Ligands by Exponential Enrichment). Their ability to bind with high affinity and specificity has opened up a wide arena of scientific applications. Aptamers have been shown to have many potential therapeutic and bioanalytical contributions such as in the treatment of diseases like cancer, cardiovascular and other major ailments. The aim of this dissertation is to study and analyze double transversion involving 4 bases in a RNA aptamer molecule: MinB4-au-flipS clamp which is a modified version of the original B4 aptamer specific for the yeast transcription factor TFIIB. The yeast TFIIB protein was purified using His-Tag purification column and Electrophoretic Mobility Shift Assays were carried out with radiolabelled αP32 ATP to study the binding ability of the modified MinB4-au-flipS clamp aptamer with the protein. The mutational versions of MinB4-au-flipS clamp aptamer named gc30-flipL-MinB4-au-flipS-clamp and cg31-flipL-MinB4-au-flipS-clamp were constructed by flipping the mfold predicted G-C base pairs at positions 16-31 and 17-30 respectively , to observe and understand the contribution of these nucleotides at these positions in binding, i.e., whether they would enhance or decrease the affinity of the aptamer for the TFIIB protein . The mutational transversions: flip of the G-C bases, were chosen so as to cause minimal disruption to the mfold proposed secondary structure of the aptamer.

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