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1.5
Diol 1
Diol 2
1.0
0.5
0.0
[Diol] M
Fig. 6 ARS competition binding assay. Diol competition curves showing normalized fluores-
cence intensity versus diol 1 and diol 2 concentration for a DBA in a physiological buffer at
neutral pH
Fig. 7 Structures of DBA 2 (a), diol 1 (b) and diol 2 (c) evaluated for binding performance in a
diol competition binding assay
The drop in fluorescence intensity as the concentration of diol titrated into the
solution increased demonstrated that diol 1 could bind to the DBA with an affinity
sufficient to release the DBA from the DBA:ARS complex. By contrast, the DBA
showed little affinity towards diol 2, which was subsequently not considered for
immobilization as an iDIOL. Based on the results of this screening process, we
generated a library of diols that, when immobilized as iDIOLs, encompassed a
range of DBA:diol and DBA:glucose binding affinities. The database of diol/iDIOL
chemical and physical properties, as they related to binding affinity, became part of
the toolbox that enabled us to screen for the optimal signaling component relative to
the desired glucose-competitive DBA binding environment.
5 Coordinated Identification of DBA:iDIOL Lead Pairs
In order to achieve identification of lead DBA:iDIOL pairs for subsequent evalua-
tion as candidate glucose sensing system components, we required a method that
would allow us to determine the relative affinities of DBA:glucose versus DBA:
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