aptamer needs to be fluorescent. EMSA and filter-binding assays can be performed
either with fluorescent or radioactive signal readout. Only label-free MST and ITC
do not need any fluorescent modification of the interaction partners.
The following table (Table 2) summarizes sample consumption, throughput, and
necessary sample pretreatment of the selected biophysical techniques.
Table 2 Consumption, throughput, and sample pretreatment of selected biophysical methods
Technique
Consumption Throughput Sample pretreatment
SPR
Immobilization via chemical
modification or direct
capturing of target epitopes
BLI
Immobilization via chemical
modification or direct
capturing of target epitopes
SwitchSENSE
Immobilization via DNA
adaptor sequence on target
MST
Fluorescent labeling via
chemical modification or
monitoring of target-intrinsic
tryptophan fluorescence
ITC
Extensive dialysis of target
and ligand into the exact
same buffer
Flow cytometry
Fluorescent labeling of the
aptamer
Fluorescence
polarization
Fluorescent labelling of the
smaller partner
EMSA
Fluorescent or radioactive
labeling of the aptamer
Filter binding
assay
Fluorescent or radioactive
labeling of the aptamer
Green indicates low consumption or high throughput, orange medium consumption or throughput,
and red high sample consumption or low throughput
Biophysical Characterization of Aptamer-Target Interactions
9