Optimization of a PS2.M-based catalytic beacon in preparation for use as a device to explore mRNA secondary structures
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Donald Franklin Patterson (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Christopher Coburn
Abstract: The DNA oligonucleotide PS2.M has been previously reported to have nanomolar
affinity for hemin. The PS2.M-hemin complex then exhibits peroxidase activity. It was
predicted that this sequence could be used in a catalytic beacon to facilitate the
development of a laboratory tool that would be effective at determining potential siRNA
target sites. The PS2.M-based beacon used for this study forms a stem-loop structure that
opens upon hybridization with a single-stranded target sequence. Once open, the PS2.M
part of the beacon can fold, locking hemin within a three level planer arrangement. Once
hemin is in place, the structure is catalytically active and can catalyze the oxidization of
the chromogenic substrate 2,2,’-azino-bis (3-ethylebenzthiazoline-6-sulphonic acid)
(ABTS) in the presence of hydrogen peroxide. Thus, binding of the beacon can be
monitored with absorbance readings at 414 nm. Although molecular beacons with a
stem-loop structure could potentially be used for this type of test, the cost would be
prohibitively expensive for most labs. Molecular beacons cost over $300 a piece, and
many of them would be needed to conduct an exploration for a suitable siRNA target site
within an mRNA. In contrast, a PS2.M-based beacon would only cost approximately $12. In general, development and optimization of a PS2.M-based catalytic beacon could
provide a more economical and improved means of establishing siRNA target site
accessibility within an mRNA. It was concluded that K+ and Mg+ are required for
folding of the PS2.M beacon. The substrate ABTS offered the highest absorbance values
at a concentration of 3.2 mM. Also, H2O2 concentrations of 3.6 mM and an extension of
the beacon stem by 10 nucleotides will result in reduced non-specific background
absorbance activity.
Optimization of a PS2.M-based catalytic beacon in preparation for use as a device to explore mRNA secondary structures
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Created on 7/1/2010
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2010
- Subjects
- Biomolecules -- Structure -- Research
- Structure-activity relationships (Biochemistry)
- Oligonucleotides
- Small interfering RNA
- Messenger RNA
- DNA-protein interactions
- RNA-protein interactions