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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://www.wcu.edu/404.asp
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.

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