A kinetically controlled platform for ligand-oligonucleotide transduction

Abstract

Ligand-oligonucleotide transduction provides the critical pathway to integrate non-nucleic acid molecules into nucleic acid circuits and nanomachines for a variety of strand-displacement related applications. Herein, a general platform is constructed to convert the signals of ligands into desired oligonucleotides through a precise kinetic control. In this design, the ligand-aptamer binding sequence with an engineered duplex stem is introduced between the toehold and displacement domains of the invading strand to regulate the strand-displacement reaction. Employing this platform, we achieve efficient transduction of both small molecules and proteins orthogonally, and more importantly, establish logical and cascading operations between different ligands for versatile transduction. Besides, this platform is capable of being directly coupled with the signal amplification systems to further enhance the transduction performance. This kinetically controlled platform presents unique features with designing simplicity and flexibility, expandable complexity and system compatibility, which may pave a broad road towards nucleic acid-based developments of sophisticated transduction networks.

Fig. 1. Kinetic control of ligand-oligonucleotide transduction.

a Structural alteration of the nucleic acid aptamer with a designed short stem. In the absence of the target ligand, the aptamer cannot form a defined structure, leading to the inability of the stem duplex. When the ligand binds to the aptamer, the stem duplex can be stably formed with assistance from the structured binding domain. b Kinetic distinction between the free and the ligand-bound invading strand. The aptamer sequence with the short stem is inserted between the toehold and displacement domains of the invading strand. In the free state, the toehold and displacement domains cannot be closely connected to function as an effective invading strand, resulting in a slow displacement reaction. When the ligand binds to the aptamer, these two domains are tightly linked together by the formation of the stem duplex, leading to a fast kinetic behavior of the strand-displacement reaction.

Fig. 2. Kinetic control of ligand-oligonucleotide transduction regulated by small molecules.

a Design of the ATP-induced strand-displacement reaction. The ATP aptamer with a designed short stem was placed between the toehold and displacement domains of the invading strand (Apt-ATP). The addition of ATP promoted the strand displacement with Rep-1. The quenched fluorescence was then recovered after the strand displacement as an indication of the invasion process. Squares and arrows drawn on DNA strands represent 5′ termini and 3′ termini, respectively. Base-pairing is shown by gray dots. b Optimization of the stem duplex of Apt-ATP. The red curve indicated the kinetic data of the reporter system in the presence of 1 mM ATP. The black curve indicated the background signal without ATP. Different designs of the stem duplexes were labeled in the corresponding panels. c Concentration-dependent kinetic performance of the ATP-induced strand-displacement reaction. The design of the stem duplex was 5′-CTT-3′. The concentrations of ATP were 2000, 1000, 500, 200, 100, 50, 20, 10, 5 and 0 μM, respectively. d Analysis of initial kinetic rates for the change of fluorescence in the presence of different concentrations of ATP. “a.u.”: arbitrary units for fluorescence. Data in c, d are presented as mean values with standard deviations (error bars) derived from three independent experiments. “N.S.”: no significant difference; **P = 0.0038, ***P = 0.0003 (unpaired t test, two-tailed P value, n = 3). e, f Representative kinetic performance of the mycotoxin ochratoxin A (OTA) and the l-tyrosinamide (Tym) induced strand displacement-based the same design. The red curves indicated the kinetic data of the reporter system in the presence of 50 μM OTA or 100 μM Tym, respectively. The black curves indicated the background signal without ligands. Source data are provided as a Source Data file.

Fig. 3. Protein-regulated kinetic control of ligand-oligonucleotide transduction.

a Design of the thrombin (Thr) induced strand displacement reaction. Thrombin promoted the strand displacement with Rep-2 with increased fluorescence signals. b Representative kinetic performance of thrombin-induced strand displacement reaction. The design of the stem duplex was 5′-GTC-3′. The red curve indicated the kinetic data of the reporter system in the presence of 200 nM thrombin. The black curve indicated the background signal without thrombin. c Concentration-dependent kinetic performance of the thrombin-induced strand-displacement reaction. The concentrations of thrombin were 500, 250, 100, 50, 25, 10, 5, 2, and 0 nM, respectively. d Analysis of initial kinetic rates for the change of fluorescence in the presence of different concentrations of thrombin. “a.u.”: arbitrary units for fluorescence. Data in c, d are presented as mean values with standard deviations (error bars) derived from three independent experiments. **P = 0.0065, ***P = 0.0006 (unpaired t test, two-tailed P value, n = 3). e Representative kinetic performance of the PDGF protein-induced strand-displacement reaction-based the same design. The red curve indicated the kinetic data of the reporter system in the presence of 50 nM PDGF. The black curve indicated the background signal without PDGF. f Orthogonal manipulation between ATP and thrombin. Independent transduction platforms for ATP and thrombin co-existed in the same system. The ATP transduction was designed to be reported by Rep-1; the thrombin transduction was designed to be reported by Rep-2. Rep-1 and Rep-2 were fully independent without any sequence correlation. The signals of Rep-1 (left panel) and Rep-2 (right panel) were the fluorescence of the FAM and TMR fluorophores, respectively. The kinetic data in presence of both 1 mM ATP and 200 nM thrombin (the A+T curve), only 1 mM ATP (the ATP curve), and only 200 nM thrombin (the Thr curve) were measured and compared along with the background signal (the Blank curve). Source data are provided as a Source Data file.

Fig. 4. Logical operations between different ligands.

a The “OR” logic gate between ATP and thrombin. ATP and thrombin were designed to promote the strand-displacement of the same reporter system. The kinetic data in presence of both 0.5 mM ATP and 100 nM thrombin (the A+T curve), 1 mM ATP (the ATP curve), and 200 nM thrombin (the Thr curve) were measured and compared along with the background signal (the Blank curve). b The “NOT” logic gate between ATP and thrombin. The ATP aptamer was designed to function as an inhibitor against thrombin transduction. The concentration of thrombin was 200 nM; the concentrations of ATP were 0, 0.2, 0.5, 1, and 2 mM, respectively. The black curve indicated the background signal without thrombin or ATP. c Utilization of combinatory aptamer to build the “AND” logic gate between ATP and thrombin. The two aptamer designs were combined to function together through the joint duplex. The kinetic data in presence of both 1 mM ATP and 200 nM thrombin (the A+T curve), only 1 mM ATP (the ATP curve), and only 200 nM thrombin (the Thr curve) were measured and compared along with the background signal (the Blank curve). Signals of all these strand-displacement reactions were reported by the Rep-2 system. Source data are provided as a Source Data file.

Fig. 5. Cascade reactions between different ligands.

a Release of the displacement domain for the cascade reaction. The addition of thrombin triggered the release of the masked displacement domain for the ATP transduction, and subsequently, the reporter system was activated by the ATP-induced strand displacement. Signals of these strand-displacement reactions were reported by the Rep-2 system. b Release of the toehold domain for the cascade reaction. The addition of thrombin triggered the release of the masked toehold domain for the ATP transduction, and subsequently, the reporter system was activated by the ATP-induced strand displacement. Given that the toehold domain was generally too short to maintain a stable duplex at room temperature, an extended strand was utilized to stably inhibit the toehold. Signals of these strand-displacement reactions were reported by the Rep-1 system. The kinetic data in presence of both 1 mM ATP and 200 nM thrombin (the T→A curve), only 1 mM ATP (the ATP curve), and only 200 nM thrombin (the Thr curve) were measured and compared along with the background signal (the Blank curve). Source data are provided as a Source Data file.

Fig. 6. Ligand-oligonucleotide transduction coupled with other nucleic acid-based techniques.

a The ATP-induced oligonucleotide output coupled with the toehold exchange-based catalysis. Once the output strand was released upon the addition of ATP, the following toehold exchange system was initiated with amplified output signals. The fluorescence change after 4-h incubation in the presence of different concentrations of ATP was shown in the columns. Signals were reported by the Rep-3 system. ***P = 0.0008 (unpaired t test, two-tailed P value, n = 3). b The ATP-induced oligonucleotide output coupled with the magnesium-based DNAzyme system. The restrained DNAzyme strand was released upon the addition of ATP. The dual-labeled substrate was cleaved by the DNAzyme with increased fluorescence signals. The fluorescence change after 1-h incubation in the presence of different concentrations of ATP was shown in the columns. ***P = 0.0003 (unpaired t test, two-tailed P value, n = 3). Data in a and b are presented as mean values with standard deviations (error bars) derived from three independent experiments. Source data are provided as a Source Data file.