Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA

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Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA
Title:
Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA
Other Titles:
Nucleic Acids Research
Keywords:
Publication Date:
08 August 2019
Citation:
Michiko Kimoto, Yun Wei Shermane Lim, Ichiro Hirao, Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA, Nucleic Acids Research, Volume 47, Issue 16, 19 September 2019, Pages 8362–8374, https://doi.org/10.1093/nar/gkz688
Abstract:
Many nucleic acid aptamers that bind to target molecules have been reported as antibody alternatives. However, while the affinities of aptamers vary widely, little is known about the relationship between the affinities and their applicabilities for practical use. Here, we developed molecular affinity rulers: a series of DNA aptamers with different affinities that bind to the same area of target molecules, to measure the aptamer and its device applicabilities. For the ruler preparation, we used high-affinity DNA aptamers containing a hydrophobic unnatural base (Ds) as the fifth base. By replacing Ds bases with A bases in Ds-DNA aptamers targeting VEGF165 and interferon-γ, we prepared two sets of DNA aptamers with dissociation constants (KD) ranging from 10-12 to 10-8 M. Using these molecular affinity rulers, we evaluated the sensitivity of DNA aptamers in ELISA (enzyme-linked immunosorbent assay), which showed the clear relationship between aptamer affinities and their detection sensitivities. In sandwich-type ELISA using combinations of aptamers and antibodies, aptamers with KD values lower than ~10-9 M were required for sufficient sensitivities (limit of detection (LOD) <10 pM) and signal intensities, but optimizations improved the lower-affinity aptamers’ applicabilities. These aptamer affinity rulers could be useful for evaluating and improving aptamer applicabilities.
License type:
http://creativecommons.org/licenses/by/4.0/
Funding Info:
This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) and National Research Foundation of Singapore (NRF-CRP17-2017-07).
Description:
ISSN:
0305-1048
1362-4962
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