A general approach to the design of allosteric, transcription factor-regulated DNAzymes

Abstract

Here we explore a general strategy for the rational design of nucleic acid catalysts that can be allosterically activated by specific nucleic-acid binding proteins. To demonstrate this we have combined a catalytic DNAzyme sequence and the consensus sequence recognized by specific transcription factors to create a construct exhibiting two low-energy conformations: a more stable conformation lacking catalytic activity and lacking the transcription factor binding site, and a less stable conformation that is both catalytically active and competent to bind the transcription factor. The presence of the target transcription factor pushes the equilibrium between these states towards the latter conformation, concomitantly activating catalysis. To demonstrate this we have designed and characterized two peroxidase-like DNAzymes whose activities are triggered upon binding either TATA binding protein or the microphthalmia-associated transcription factor. Our approach augments the current tool kit for the allosteric control of DNAzymes and ribozymes and, because transcription factors control many key biological functions, could have important clinical and diagnostic applications.

Fig. 1. Transcription factor-induced activation of a DNAzyme. Here we demonstrate a DNAzyme allosterically activated by specific transcription factors (TF-regulated DNAzyme). To do so we coupled two functional domains: (i) a catalytic DNAzyme domain (red sequence in the cartoon) and (ii) a double-stranded transcription factor (TF)-binding domain (green). A sequence element complementary to the sequence of the DNAzyme stabilizes an alternative conformation (left) that “sequesters” both domains in an inactive (i.e., non-catalytic and non-TF-binding, respectively) state. This off-state is in equilibrium with a second conformation, the on-state, in which both domains are functional. TF binding shifts this equilibrium towards the on-state, activating catalysis. Here we used the HRP-like G-quadruplex DNAzyme as our model catalytic domain. In the presence of hemin and hydrogen peroxide, this domain catalyzes the oxidation of the HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to give a coloured product which is detectable by absorbance (λ_max = 650 nm).

Fig. 2. As a first proof-of-principle of transcription factor-induced DNAzyme activation, we designed four DNAzyme variants that are activated by MITF, a transcription factor that is involved in cancer progression. (A) Initially, in order to characterize the different variants, we used a simple DNA strand mimicking the action of the target TF (mimicking strand). (B) In order to modulate the range of the effector's concentration at which activation is observed, the variants were designed with increasingly stable off-states whilst maintaining approximately constant stability for the on-state (indicated are the m-fold predicted free energies). (C) As expected, the concentration range over which the allosteric effector causes activation of the DNAzyme is dependent on the stability of the off-state. (D) Optimal DNAzyme activation is observed at intermediate stabilities of the off-state (the y-axis shows the DNAzyme activation, the difference in activity when changing the effector concentration from 10 nM to 1 μm). DNAzyme activation was followed here and in the next figures by measuring absorbance at 650 nm in a solution of 50 nM TF-regulated DNAzyme in the presence of the DNAzyme cofactor hemin (500 nM) 40 min after the addition of the DNAzyme substrates TMB and H₂O₂. For clarity, error bars have been depicted for only the last point on these binding curves and those in the following figures. These error bars represent the standard deviations of measurements performed on at least three independent replicates.

Fig. 3. (Left) Our MITF-regulated DNAzyme (variant #2) is activated by its target TF in a dose-dependent fashion and is insensitive to other, non-targeted proteins (right). For the specificity test (right) we have used a concentration of target MITF and of non-specific proteins of 500 nM. In the y-axis of the right panel the difference (Δabsorbance) between the absorbance value obtained in the presence of the target and that obtained in a blank solution has been used.

Fig. 4. (A) To demonstrate the generality of our strategy we designed variants containing the consensus binding site for TATA binding protein (TBP). (B) Using a DNA strand mimicking the action of the target TF (see Fig. 2a), we find that, as expected, increasing the off-state stability shifts the activation profile to higher effector concentrations. (C) The intermediate variant (variant #2) was selected for further tests with the TF showing the activation of the TF-regulated DNAzyme as the concentration of TBP increases (EC₅₀ = 104 ± 12 nM). (D) This DNAzyme, too, exhibits good specificity against other non-specific proteins. Here, we have used a concentration of target TBP and of non-specific proteins of 500 nM. In the y-axis of panel D the difference (ΔAbsorbance) between the absorbance value obtained in the presence of the target and that obtained in a blank solution has been used.