Aptamer-Based DNA Allosteric Switch for Regulation of Protein Activity

Abstract

Allostery is a fundamental way to regulate the function of biomolecules playing crucial roles in cell metabolism and proliferation and is deemed the second secret of life. Given the limited understanding of the structure of natural allosteric molecules, the development of artificial allosteric molecules brings a huge opportunity to transform the allosteric mechanism into practical applications. In this study, the concept of bionics is introduced into the design of artificial allosteric molecules and an allosteric DNA switch with an activity site and an allosteric site based on two aptamers for selective inhibition of thrombin activity. Compared with the single aptamer, the allosteric switch possesses a significantly enhanced inhibition ability, which can be precisely regulated by converting the switch states. Moreover, the dynamic allosteric switch is further subjected to the control of the DNA threshold circuit for realizing automatic concentration determination and activity inhibition of thrombin. These compelling results confirm that this allosteric switch equipped with self-sensing and information-processing modules puts a new slant on the research of allosteric mechanisms and further application of allosteric tactics in chemical and biomedical fields.

Keywords: allostery; aptamer; bionics; enzyme activity regulation; molecular switch.

Scheme 1

Schematic illustration of the designed aptamers‐based allosteric switch. a) The bionic allosteric switch, in which poly A linker is the allosteric site, and the entirety of two aptamers is the active site. b) The conformation and function conversion of the allosteric switch is controlled by the TMSD reaction due to the addition of the allosteric effector (AA) and allosteric antidote (AE). c) The allosteric switch is plugged into the DNA threshold circuit for the logic operation to realize the selective inhibition of thrombin activity.

Figure 1

Optimization of poly A linker length in the synthetic allosteric switch. a) The spatial position of HD1 and HD22 binding to thrombin, respectively. HD1 binds to the fibrinogen‐recognition exosite (exosite I), while HD22 binds to the heparin‐binding exosite (exosite II). b) The linker length of the allosteric switch affects the binding of HD1 and HD22 to thrombin. The longer linker causes the allosteric switch to extend further, while the suitable linker endows the allosteric switch with a more powerful inhibition effect of thrombin activity. c) The kinetics of light scattering due to the conversion of fibrinogen (1.25 mg mL⁻¹) into fibrin catalyzed by thrombin (0.75 U mL⁻¹, Control) or thrombin that were preincubated with different allosteric switches (10 nm, ASnA, n = 10, 20, and 30). The fill area under the solid lines is the error bar (n = 3, mean ± SD). d) The relative activity of thrombin that was preincubated with different allosteric switches (n = 3, mean ± SD). ns, p > 0.05; ^*** p < 0.001.

Figure 2

Enhanced ability of the allosteric switch to inhibit thrombin activity compared with HD1. a) Relative activity of thrombin that was preincubated with different concentrations of HD1 or AS20A (n = 3, mean ± SD). With the increase of AS20A from b) 0.1 to 5 nm or HD1 from c) 5 to 100 nm, the relative activity of thrombin (0.75 U mL⁻¹) decreases obviously. Compared with HD1, AS20A has a lower IC50 (the corresponding allosteric effector concentration when the relative activity is 50%) and a narrower concentration window.

Figure 3

The reversible conversion of conformation and function of AS20A is realized by adding AE and AA. a) Scheme illustration of the conversion process of the allosteric switch. b) PAGE image showing the reversible state conversion of AS20A (lane 1: AS20A; lane 2: AS20A‐AE; lane 3: AS20A‐AE/AA; lane 4: 20 bp DAN ladder). c) The inhibition rate of the allosteric switches in different states to thrombin activity (n = 3, mean ± SD). d) The reversible inhibition of the allosteric switch (“on” state: green dots, “off” state: red dots) by alternately adding AE and AA to AS20A for three cycles (n = 3, mean ± SD). ns, p > 0.05; ^*** p < 0.001.

Scheme 2

Schematic illustration of the integrated allosteric circuit in which the allosteric switch is plugged into the DNA threshold circuit for selective inhibition of thrombin activity. Part 1: The allosteric switch recognizes thrombin through aptamer (HD22) and releases INPUT strand of corresponding quantities. Part 2: The threshold controller determines whether INPUT released in the previous part is sufficient to start the entropy‐driven circuit. Part 3: INPUT passed through part 2 is converted and accessed into the signal amplifier, and then amplified into a large number of AA to execute the conformation and function conversion of the allosteric switch. Insert: The flowchart of the integrated allosteric circuit.

Figure 4

Research on the feasibility of constructing the integrated allosteric circuit step by step. a) Schematic illustration of the three‐step process of target identification, threshold, and signal amplification. b) The nonlinear fitting curve of fluorescence (n = 3, mean ± SD). HD22 in the IAA complex recognizes and binds thrombin to induce INPUT release and fluorescence signal recovery. c) Calibration curve of fluorescence intensity and INPUT concentration (n = 3, mean ± SD). d) Response of threshold controller to different intensities of INPUT (n = 3, mean ± SD). The entropy‐driven signal amplifier powered by FUEL enables H2 to cause e) faster and f) more significant fluorescence recovery (n = 3, mean ± SD). g) The flowchart of conformation and function conversion of allosteric switch initiated by INPUT through DNA threshold and signal amplifier. h) DNA circuits with and without threshold respond to normal‐level INPUT and high‐level INPUT (n = 3, mean ± SD): i) normal‐level INPUT + DNA circuits with threshold; ii) normal‐level INPUT + DNA circuits without threshold; iii) high‐level INPUT + DNA circuits with threshold; iv) high‐level INPUT + DNA circuits without threshold. Normal‐level INPUT: 9.383 nm; high‐level INPUT: 21.64 nm; DNA circuits with threshold: 12.5 nm NLP, 50 nm HLP, 50 nm A1‐AA, 100 nm FUEL, and 50 nm AS20A_FAM‐AE_BHQ1; DNA circuits without threshold: 50 nm HLP, 50 nm A1‐AA, 100 nm FUEL, and 50 nm AS20A_FAM‐AE_BHQ1.

Figure 5

The integrated allosteric circuit differentiates the normal‐level and high‐level thrombin and inhibits thrombin activity selectively. a) Schematic illustration of the integrated allosteric circuit response to different levels of thrombin. b) Measurements of thrombin‐catalyzed cleavage of fibrinogen (1.25 mg mL⁻¹) under different concentration and incubation conditions. Normal‐level thrombin: 3.75 U mL⁻¹ (red series); high‐level thrombin: 11.25 U mL⁻¹ (blue series). i) control: 50 nm IAA; ii) integrated allosteric circuit with threshold: 50 nm IAA, 12.5 nm NLP, 50 nm HLP, 50 nm A1‐AA, 100 nm FUEL; iii) integrated allosteric circuit without threshold: 50 nm IAA, 50 nm HLP, 50 nm A1‐AA, 100 nm FUEL. c) Heat map and d) histogram of the relative activity of thrombin at normal and high levels after incubation with two kinds of DNA circuit (n = 3, mean ± SD).