DNA Logic Gate Based on Metallo-Toehold Strand Displacement

<div><p>DNA is increasingly being used as an ideal material for the construction of nanoscale structures, circuits, and machines. Toehold-mediated DNA strand displacement reactions play a very important role in these enzyme-free constructions. In this study, the concept of metallo-toehold was utilized to further develop a mechanism for strand displacement driven by Ag⁺ ions, in which the intercalation of cytosine–cytosine mismatched base pairs on the toeholds provides additional control by varying of the concentration of Ag⁺ ions. The characteristics of displacement reaction in response to different concentration of Ag⁺ ions are investigated by fluorescence spectral and non-denaturing polyacrylamide gel electrophoresis. The reaction can successfully occur when the concentration of Ag⁺ ions is suitabe; excess Ag⁺ ions block the reaction. Furthermore, the displacement reaction can be tuned and controlled most efficiently under the condition of two C:C mismatched base pairs placed on the six-nt toehold. Based on our research, a mechanism was developed to construct Boolean logic gate AND and OR by employing strand displacement reaction as a tool, Ag⁺ and Hg²⁺ as input.</p></div

The association of I-CaM with LSEL15 in aqueous solution.

<p>A, Trp fluorescence emission spectra of 1 nM LSEL15 in the absence (solid line) and presence (dash line) of 10 nM Ca²⁺-loaded CaM. LSEL15 and CaM were dissolved in 2 ml of 10 mM MOPS, 0.3 mM CaCl₂, 100 mM NaCl, 0.1 mg/ml BSA, pH 7.4. The emission spectra were obtained with the excitation wavelength at 295 nm and each was the average of 3 scans. Both spectra were corrected for background fluorescence from the buffer. B, the titration plot of LSEL15 by Ca²⁺-CaM. CaM was titrated to the LSEL15 solution at indicated concentrations, and the Trp emission at 333 nm was measured. Dashed line indicates the fitted binding curve. C, IAEDANS fluorescence emission spectra of 1 nM Ca²⁺-I-CaM in the absence (solid line) and presence (dash line) of 25 nM LSEL15. I-CaM and LSEL15 were dissolved in 2 ml of the same buffer as in (A). The emission spectra were obtained with the excitation wavelength at 340 nm and each was the average of 3 scans. Both spectra were corrected for background fluorescence from the buffer. D, the titration plot of Ca²⁺ loaded I-CaM by LSEL15. LSEL15 was titrated to the I-CaM solution at indicated concentration, and the IAEDANS emission at 475 nm was measured. Dashed line indicates the fitted binding curve.</p

Fluorescence spectra of the reactions after treatment with Ag⁺ ions for 24 h.

<p>Final concentration: [target complex] = 100 nM; [fluorescence reporter complex] = 150 nM; [Input-oligomer] = 400 nM. The number in the figure represented the ratio [Ag⁺]/[Target complex]. (a) Target complex is consist of Signal and S5-1, 3; In5-1,3 severs as Input-oligomer. (b) Target complex is consist of Signal and S7-1,3; In7-1,3 severs as Input-oligomer. (c) The completely complementary strand of substrate severs as input-oligomer, all other chains are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111650#pone-0111650-g001" target="_blank">Fig. 1</a>.</p

The ‘AND’ logic gate system using Hg²⁺ and Ag⁺ as inputs while the enhancement of fluorescence intensity as output.

<p>(a) The design of the logic gate. (b) The truth table of the logic gate. (c) The resultant fluorescence spectra of different input states. (d) Values of (F–F₀)/F₀ respond to different input states.</p

Schematic representation of DNA strand displacement triggered by Ag⁺ ions and fluorosence measurements strategy.

<p>F denotes ROX fluorophore, Q denotes BHQ-2 quencher. The reporter complex reacts stoichiometrically with the product Signal to induce an increase in fluorescence intensity.</p

Sequences of L-selectin-derived peptides CLS and LSEL15.

<p>The sequence of C-terminal portion of human L-selectin, including entire transmembrane and cytoplasmic domains, is shown on top, with key residue numbers included. Residues in the transmembrane domain are underlined. The filled triangle marks the shedding cleavage site in L-selectin. The N-terminal end of LSEL15 is acetylated (ac-).</p

Fluorescence spectra of the reactions after treatment with Ag⁺ ions for 24 h.

<p>Final concentration: [target complex] = 100 nM; [fluorescence reporter complex] = 150 nM. Target complex consists of Signal and S5-1,3. (a) [In5-1 m] = 400 nM. (b) [In5-3 m] = 400 nM. The number in the figure represented the ratio [Ag⁺]/[Target complex]. (c) Gel electrophoresis images of strand displacement reaction triggered by Ag⁺ ions, obtained after 24 h. Except for the lane M, that first 4 lanes correspond to In5-1 m and the last four correspond to In5-3 m respectively. The number above each lane represents the relative concentration of Ag⁺ ions to the target complex.</p

CaM adopts an extended conformation in its ternary complex with CLS and moesin FERM domain.

<p>A, time-based IAEDANS emission traces of 125 nM I-CaM in 10 mM MOPS, 100 mM NaCl, 0.3 mM CaCl₂, pH 7.4. 10 µM (final concentration) moesin FERM domain or 10 µM (final concentration) CLS/POPC/POPS (1/850/150 molar ratio) or empty POPC/POPS liposome was added to the I-CaM solution at indicated time points. Differently colored traces indicate separate runs. The excitation and emission wavelengths were set to 340 and 475 nm, respectively. B, The titration plot of I-CaM by the complex of CLS/POPC/POPS and moesin FERM domain. The mixture of CLS/POPC/POPS and moesin FERM, at 1∶1 molar ratio, was titrated to the I-CaM solution at indicated concentrations, and the IAEDANS emission at 475 nm was measured. The background scattering fluorescence as a result of added liposome was subtracted from each measurement. Dashed line indicates the fitted binding curve. C, IAEDANS fluorescence emission spectra of 30 nM D/A-CaM in the buffer (○), of 30 nM D/A-CaM mixed with empty POPC/POPS liposome (□), and of 30 nM D/A-CaM mixed with 10 µM CLS/POPC/POPS (1/850/150 molar ratio) and 10 µM moesin FERM domain (•). All emission spectra were obtained with the excitation wavelength at 340 nm and each was the average of 3 scans. All spectra were corrected for background fluorescence from the buffer.</p

Differential FRET between L-selectin fragments and CaM in membrane and aqueous conditions.

<p>A, a ribbon diagram showing the proximity of Trp³¹⁵ of LSEL15 and Lys⁷⁵ of CaM in the CaM/LSEL15 complex structure (PDB ID: 2LGF). The LSEL15 peptide is shown in green, and CaM in purple. Four calcium ions are shown as yellow spheres. The side chains of Trp³¹⁵ and Lys⁷⁵ are shown in sticks, with the dashed line connecting the Nε1 atom in Trp³¹⁵ and the Nζ atom in Lys⁷⁵. B, Trp emission spectra of 26 nM LSEL15 mixed with 104 nM unlabeled CaM in 10 mM MOPS, 100 mM NaCl, 0.3 mM CaCl₂, pH 7.4 (▵), of 104 nM I-CaM alone in the same buffer (○) and of 26 nM LSEL15 mixed with 104 nM I-CaM in the same buffer (▴). C, Trp emission spectra of 26 nM CLS/POPC (1/1000 molar ratio) mixed with 104 nM unlabeled CaM in the same buffer as in (B) (▵), of 10 µM I-CaM alone with empty POPC liposome (○), and of 26 nM CLS/POPC mixed with 10 µM I-CaM (▴). All emission spectra were obtained with the excitation wavelength at 295 nm and each was the average of 3 scans. All spectra were corrected for background fluorescence from the buffer.</p

Fluorescence spectra of the reactions after treatment with Ag⁺ ions for 24 h.

<p>Final concentration: [Target complex] = 100 nM; [Fluorescence reporter complex] = 150 nM; [Input-oligomer] = 400 nM. The number in the figure represented the ratio [Ag⁺]/[Target complex].</p