ππ― π·πͺπ΅π³π° selection has been widely used to isolate single-stranded DNA molecules from large random-sequence pools that are able to perform a desired catalytic reaction, or bind to a target molecule. Using this technique, we have created a DNA enzyme, named DET22-18, which has a uniquely linked chemical catalysis/real-time signaling capability. It is a true enzyme with a π¬cββ of~7 minβ»ΒΉ -the second fastest rate ever reported for a DNA enzyme. The DNA enzyme cleaves a substrate containing a single ribonucleotide linkage embedded in a DNA chain and sandwiched between a fluorophore-labeled deoxyribonucleotide and a quencher-modified deoxyribonucleotide. The ability of DET22-18 to generate a large fluorescence signal provides a useful tool to engineer potential allosteric deoxyribozyme biosensors for real-time detection of important biological targets. To provide a proof of concept that a reporter system can be built from the above signaling DNA enzyme, the cis-acting version of this enzyme, DEC22-18A, was engineered into an allosterically regulated deoxyribozyme biosensor that can report ATP. A preliminary investigation was also conducted to determine a possible secondary structure of the DNA enzyme. This study lays a foundation for pursuing novel signaling DNA enzymes for biological detection directed applications. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22784 |
| Date | 09 1900 |
| Creators | Mei, Shirley |
| Contributors | Li, Yingfu, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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