DNA branch migration reactions through photocontrollable toehold formation

Abstract

Strand displacement cascades are commonly used to make dynamically assembled structures. Particularly, the concept of "toehold-mediated DNA branch migration reactions" has attracted considerable attention in relation to dynamic DNA nanostructures. However, it is a challenge to obtain and control the formation of pure 1:1 ratio DNA duplexes with toehold structures. Here, for the first time, we report a photocontrolled toehold formation method, which is based on the photocleavage of 2-nitrobenzyl linker-embedded DNA hairpin precursor structures. UV light irradiation (λ ≈ 365 nm) of solutions containing these DNA hairpin structures causes the complete cleavage of the nitrobenzyl linker, and pure 1:1 DNA duplexes with toehold structures are easily formed. Our experimental results indicate that the amount of toehold can be controlled by simply changing the dose of UV irradiation and that the resulting toehold structures can be used for subsequent toehold-mediated DNA branch migration reactions, e.g., DNA hybridization chain reactions. This newly established method will find broad application in the construction of light-powered, controllable, and dynamic DNA nanostructures or large-scale DNA circuits.

Figure 1

Photocleavage and photocontrolled toehold formation system. a) The chemical structure and principle of photocleavage of PC linker-connected DNA strands (shown as unannealed). b) Annealed linked DNA hairpin precursor, irradiated at 365 nm to form DNA duplex with toehold T.

Figure 2

Photocontrolled toehold formation for toehold-mediated DNA branch migration reaction. a) The principle of photocontrolled hidden-toehold activation. b) Fluorescence test of toehold-mediated DNA branch migration reaction with different irradiation times. In a typical experiment, PC-linker-modified DNA hairpin precursor in buffer solution (150 μL, 200 nM) was irradiated with 365 nm light for different times. Then, 99 μL of photoirradiated hairpin was placed in a cuvette, and the invading strand (1 μL, 20 μM) was added to initiate the branch migration reaction. c) Plot of photocleavage fraction versus UV irradiation time. The arrow in Fig. 2 b shows the time that the invading strand was added.

Figure 3

Standard calibration curve. Plot of fluorescence intensity of released strand a versus initial concentration of duplex with exposed toehold T [slope = (2.40±0.05) × 10⁴; y-intercept = (1.29±0.47) × 10⁵; r² = 0.99843]. The reaction buffer contains 20 mM Tris-HCl, 5 mM MgCl₂ and 300 mM NaCl, pH = 7.5. This experiment was repeated three times. The small black circle on the standard curve corresponds to 92% release of hidden toehold after 20 min irradiation.

Figure 4

Native PAGE (20%) analysis of the branch migration reaction after UV irradiation for different times. In a typical experiment, photoirradiated PC-linker-modified DNA hairpin precursor (10 μM) was mixed with the invading strand (10 μM) and incubated at room temperature for 30 min. Lane 1: photoirradiated (20 min) PC-linker-modified DNA hairpin; lane 2: the invading strand; lane 3: PC-linker-modified DNA hairpin precursor (no irradiation); lane 4–8: PC-linker-modified DNA hairpin precursor irradiated for different times and incubation afterwards with the invading strand.

Figure 5

Photocontrolled toehold formation for DNA hybridization chain reaction. a,b) Principle of photocontrolled toehold formation for DNA hybridization chain reaction (HCR). c) Native PAGE (10%) analysis of the photocontrolled HCR. In a typical experiment, after different photoirradiation times, H3 (6 μM) was mixed with H1 (12 μM) and H2 (12 μM) and incubated at room temperature overnight. Lane 1: H3; lane 2: H3 mixed with H1 and H2 (no irradiation); lanes 3 to 8: H3 photoirradiated for different times and mixed with H1 and H2.

Figure 6

Kinetics of photocontrolled DNA hybridization chain reaction system. a) Experimental design. In a typical experiment, a mixture of Pyrene-H1 (45 μL, 3 μM) and Pyrene-H2 (45 μL, 3 μM) were placed in a cuvette, and then photoirradiated H3 (15 μL, 3 μM) was added to initiate the reaction. b) Plot of excimer emission intensity versus polymerization reaction time for different H3 irradiation times. The fluorescence intensities at 475 nm were recorded at different times (λ_ex = 340 nm).