@article{8e7c63e4592749f191b39d76ba32168c,
title = "Idiosyncrasies of thermofluorimetric aptamer binding assays",
abstract = "To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer-target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.",
keywords = "Aptamer, Binding assay, Cell-SELEX, EGFR, EvaGreen, Hybrid fluorescence, Melt curve analysis, Open qPCR, SELEX, Thermofluorimetric analysis",
author = "Damase, {Tulsi Ram} and Allen, {Peter B.}",
note = "Funding Information: This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM104420. This published report is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P30 GM103324. The content is solely the responsibility of the authors and does not necessarily represent the o ?cial views of the National Institutes of Health. The authors have no other relevant a ?lia-tions or financial involvement with any organization or entity with a ?nancial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Funding Information: This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM104420. This published report is supported by an Institutional Devel-opment Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P30 GM103324. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Publisher Copyright: {\textcopyright} 2019 Peter B Allen",
year = "2019",
month = mar,
day = "1",
doi = "10.2144/btn-2018-0128",
language = "English (US)",
volume = "66",
pages = "121--127",
journal = "BioTechniques",
issn = "0736-6205",
publisher = "Eaton Publishing Company",
number = "3",
}