Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy

Abstract

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF165 was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF165 was dependent on the concentration of VEGF165. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF165 complexes was revealed, which was similar to that of the intact aptamer/VEGF165. Besides, the dissociation rate constant (koff) of split aptamer/VEGF165 was close to that of intact aptamer/VEGF165, and the interaction force of split aptamer/VEGF165 was higher than the force of intact aptamer/VEGF165. It indicated that split aptamer also possessed high affinity with VEGF165. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level.

Engineering and Application of a Myoglobin Binding Split Aptamer

“…Since the binding mechanism between the intact aptamers and Myo has not been clarified, the intact aptamer against Myo was first randomly divided at different sites into split aptamer 1 and split aptamer 2, as shown in Scheme . Then, split aptamer 1 and split aptamer 2 were attached to the AFM tip and gold substrate, respectively. , Next, AFM measurements were carried out in the absence or presence of Myo . Given that there was a significant difference in the binding probability with or without the target, it was speculated that the binding probability may be one of the judgment parameters for engineering split aptamer.…”

“…30,31 Next, AFM measurements were carried out in the absence or presence of Myo. 29 Given that there was a significant difference in the binding probability with or without the target, it was speculated that the binding probability may be one of the judgment parameters for engineering split aptamer. That is, if the binding probability was high, it was suggested that the split aptamers could bind to the target.…”

“…The interaction between split aptamers and Myo was measured using AFM (JPK Instruments, Germany). Similar to our previous work, one of the split aptamers was modified on the AFM tip, and another split aptamer was functionalized on the gold substrate. As mentioned in our previous works, , the intact aptamer was attached to the tip, Myo was adsorbed on the gold substrate, and the interaction between aptamers and Myo was measured.…”

Exploring Interactions of Aptamers with Aβ40 Amyloid Aggregates and Its Application: Detection of Amyloid Aggregates

“…The force measurements between Apt1- or Apt2-functionalized tips and Aβ 40 -aggregate-modified Au substrates were conducted at a loading rate of 3.52 × 10 4 pN/s by JPK biological AFM (JPK Instruments AG, Germany), which were shown in Figure A. Herein, the utility of PEG 3400 linker could raise the steric flexibility and separate different dissociation events. , Further, in order to obtain a single dissociation event, several experiment conditions, including immobilization density of Apts and Aβ 40 aggregates, were optimized. , Simultaneously, a series of control groups were performed to distinguish between specific and nonspecific interactions. As shown in Figure B, the specific peak would vanish when Apts-functionalized AFM tips were blocked with Aβ 40 aggregates or the Aβ 40 -aggregate-modified Au substrates were blocked with Apts.…”

“…Herein, the utility of PEG 3400 linker could raise the steric flexibility and separate different dissociation events. 29,30 Further, in order to obtain a single dissociation event, several experiment conditions, including immobilization density of Apts and Aβ 40 aggregates, were optimized. 31,32 Simultaneously, a series of control groups were performed to distinguish between specific and nonspecific interactions.…”