An aptamer beacon responsive to botulinum toxins
| Autor: | Maria P. Carrillo, Allison Edge, Alicia M. Richarte, John G. Bruno |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Botulinum Toxins
Aptamer Biomedical Engineering Biophysics Spectrofluorometer Molecular Probe Techniques Reproducibility of Results General Medicine Biosensing Techniques Equipment Design Biology Aptamers Nucleotide Immunoglobulin light chain Fluorescence Molecular biology Sensitivity and Specificity DNA sequencing Equipment Failure Analysis Fluorometer Electrochemistry Function (biology) Systematic evolution of ligands by exponential enrichment Biotechnology |
| Zdroj: | Biosensorsbioelectronics. 31(1) |
| ISSN: | 1873-4235 |
| Popis: | Sixty candidate DNA aptamers were developed against botulinum neurotoxin (BoNT) type A light chain (LC) from ten rounds of selection, resulting in several identical sequences. Secondary structures of the identical aptamers were compared to structures of previously reported BoNT A DNA aptamers. A series of ten candidate loop structures were selected from this comparison as potential binding pockets and aptamer beacons. These candidate beacons were synthesized with 5'-TYE 665 and 3'-Iowa Black quencher labels for comparison of fluorescence levels as a function of BoNT A LC concentration. Only three of the ten candidates exhibited any fluorescence response to increasing levels of BoNT A LC. However, of the two most responsive candidates, one represented a subset loop of the larger more intensely fluorescent double-looped structure, designated Beacon 10. This beacon yielded a lower limit of detection of 1 ng/mL in buffer using a spectrofluorometer and a portable handheld fluorometer, but also responded substantially to BoNT A, B, E holotoxins and heavy or light chain components even in a dilute soil suspension, but not in 50% human serum. Beacon 10 did not respond strongly to a variety of other divergent peptides, suggesting that it is relatively specific to the level of botulinum toxins and is only useful for environmental testing. Beacon 10 also shared short sequence segments with other published BoNT aptamer DNA sequences, suggesting that these may be points of physical contact between the aptamers and BoNTs. |
| Databáze: | OpenAIRE |
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