Kinetics of signaling-DNA-aptamer–ATP binding

DNA aptamers are molecular biosensors consisting of single functionalized DNA molecules, which can bind to specific targets or complementary DNA sequences. The binding kinetics of DNA aptamers is studied by fluorescence quenching at $23°\text{C}$. A kinetic model for the binding reaction of DNA aptamer, antisense DNA, and ATP target is developed to describe experimental observations. The approach leads to a simple procedure to deduce relevant kinetic reactions and their rate constants. A comparison between theory and experiments indicates that the previously established bimolecular DNA-ATP binding does not provide a complete description of the experimental data. Side reactions such as trimolecular complexation are proposed. Rate constants of the model are determined by comparing the model predictions and experiments. Good agreements between the model and experiments have been obtained. Possible blocking reactions by the misfolded DNA aptamer are also discussed.

Figure 1

The schematic description for the binding reaction of DNA aptamer, QDNA (quencher), and ATP target. When the DNA aptamer $\left(F\right)$ and the quencher $\left(Q\right)$ are dissociated, the fluorescence arises (lower left and upper right). The binding between the DNA aptamer and the quencher induces a large decrease in the fluorescence intensity (lower right and upper left). The lower right figure shows that the quencher binds to the DNA aptamer-ATP complex.

Figure 2

ATP-binding DNA aptamer and antisence DNA as a model system. Q10, Q11, Q12, and Q13 denote, respectively, the number of base sequences. For example, Q10 has 10 base sequences.

Figure 3

The dissociation rate of $FQ$, $k_1$, as a function of $N$. The line denotes the linear regression of three points for Q10, Q11, and Q12.

Figure 4

Normalized fluorescence intensity for Q10 in the reaction of $F+Q$ at $23°\text{C}$. The dotted lines and solid line correspond to the five-repeated experiments and theoretical result, respectively.

Figure 5

Normalized fluorescence intensity for Q11 in the reaction of $F+Q$ at $23°\text{C}$. The dotted lines and solid line correspond to the five-repeated experiments and theoretical result, respectively.

Figure 6

Normalized fluorescence intensity for Q12 in the reaction of $F+Q$ at $23°\text{C}$. The dotted lines and solid line correspond to the five-repeated experiments and theoretical result, respectively.

Figure 7

Normalized fluorescence intensity for Q13 in the reaction of $F+Q$ at $23°\text{C}$. The dotted lines and solid line correspond to the five-repeated experiments and theoretical result, respectively.

Figure 8

Comparison of the experimental normalized fluorescence intensities for Q13 and Q10 at $23°\text{C}$ to the calculated results. The theoretical result with a solid line is obtained by a set of parameters determined by minimizing the least-squares difference. The five dotted lines for Q13 denote, respectively, the five-repeated experiments in the same condition that QDNA is added to the solution of the DNA aptamer and ATP target. The parameters for Q10 at $23°\text{C}$ which we determined in the condition, ${\left[F\right]}_0=20\text{nM}$, ${\left[Q\right]}_0=40\text{nM}$, and ${\left[T\right]}_0=0.75\text{mM}$, were employed to calculate the normalized fluorescence intensity for Q10 at $23°\text{C}$ with the different concentration, ${\left[F\right]}_0=60\text{nM}$, ${\left[Q\right]}_0=30\text{nM}$, and ${\left[T\right]}_0=0.9\text{mM}$.

Figure 9

Time development for the concentration of each reactant scaled by ${\left[F\right]}_0$. The calculation was performed with ${\left[F\right]}_0=20$ nM, ${\left[Q\right]}_0=40\text{nM}$, and ${\left[T\right]}_0=0.75\text{mM}$. The concentration of the ATP target is not shown in the figure. As seen in the figure, the production of $\left(F\right)$ and $\left(FQ\right)$ are relatively small during the reaction.