Supporting Information. DNA Tetraplexes-Based Toehold Activation for Controllable DNA Strand Displacement Reactions

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1 Supporting Information DNA Tetraplexes-Based Toehold Activation for Controllable DNA Strand Displacement Reactions Wei Tang, Huaming Wang, Dingzhong Wang, Yan Zhao, Na Li, and Feng Liu* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing , China * Corresponding author. liufeng@pku.edu.cn Text S1: Experimental Section Materials and Reagents All HPLC-purified DNA oligonucleotides were purchased from Sangon Inc. (Shanghai, China). The DNA sequences and modifications are listed in Table S1 S3. The DNA samples were dissolved in TE buffer (50 mm Tris(hydroxymethyl)metyl aminomethane (Tris), 1 mm ethylene diamine tetraacetic acid (EDTA); ph 8.0) and stored in the dark at 4 o C. Adenosine triphosphate (ATP), thymine triphosphate (TTP), cytosine triphosphate (CTP), and guanosine triphosphate (GTP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Strontium chloride (SrCl 2 ) and all other chemicals employed were of analytical grade from Beijing Chemical Works (Beijing, China), and deionized water was used in all experiments. Fluorescence Measurements For preparation of SR duplex, strand S was mixed with strand R labeled by the 5 -TET and the 3 -TAMRA at 1:1 ratio in TNM buffer (50 mm Tris, 100 mm NaCl and 5 mm MgCl 2 ; ph 7.5) with a final concentration of 1 µm for each strand. The resulting solutions were annealed by heating at 95 C for 5 min and then slowly cooled down to room temperature over 2 hours. Fluorescence measurements were performed by using an F-7000 fluorescence spectrometry (Hitachi, Japan) at 25 C. Sample solutions were excited at 522 nm, and the S1

2 emission signal was recorded with wavelength of 539 nm. For all time-dependent fluorescence tests, appropriate volumes of DNA stock solutions were added to TNM buffer to achieve 20 nm final concentration with a total volume of 1.0 ml in the cuvettes. Afterwards, corresponding concentration of Sr 2+ and strand I G, with 2 µl and 1 µl of volume, respectively, were added respectively and mixed quickly within 30 s. In all graphs, time t = 0 indicates the time of strand I G being added to the solutions. Native Polyacrylamide Gel Electrophoresis Experiments In a typical experiment, the reaction mixture contained 2 µm S G R duplex, 2 µm strand I G, and 10 mm Sr 2+ was incubated at room temperature for 30 min. 12% native polyacrylamide gel electrophoresis (PAGE) experiments were carried out at 15 V/cm in 1 TBE buffer (90 mm Tris, 90 mm boric acid, 2 mm EDTA; ph 8.0) for 2 hours. After separation, PAGE gels containing DNA were stained using GelSafe Dye, and imaged by a Tanon 1600 imager (Tanon, China). Circular Dichroism Measurements Circular dichroism (CD) spectra were recorded on a Jasco-815 CD spectrometer (JASCO, Japan), using a quartz cell of 5 mm optical path length. For the CD experiment, the sample with a mixture of 0.5 µm S G R duplex, 0.5 µm strand I G, and 10 mm Sr 2+ was prepared and incubated at room temperature for 30 min. Three scans from 230 nm to 340 nm at 25 C were accumulated and averaged. The background of the buffer solution was subtracted from the CD data. Text S2: Fluorescence Data Processing The 27 nt strand R is labeled with a TET fluorophore (F) at 5 end and a TAMRA quencher (Q) at 3 end. When strand R hybridizes with strand S forming the SR duplex, the fluorophore and the quencher are separated by 26 base pair dsdna (approximately 9 nm). When strand R is displaced from the SR duplex, a random coiling of the single-stranded DNA and hydrophobic interactions bring the TET and the TAMRA sufficiently close to each other that the fluorescence of TET is almost completely quenched. 1 The strand displacement kinetics can therefore be monitored by measuring the fluorescence intensity of strand R as a function of time. The fluorescence signals are normalized using the equation S2

3 where F S is the fluorescence intensity of each sample, F R is the fluorescence intensity of strand R alone, and F SR is the fluorescence intensity of SR duplex. F R and F SR are measured before the beginning of each run as the ingredients are successively added. We presume that the proposed toehold-mediated strand displacement based on the formation of DNA tetraplex is the same as the standard toehold-based strand displacement as a simple bimolecular reaction, 1 4 (1) According to the literature, 1 consider the case when the initial concentration of invading strand I is equal to the initial concentration of SR duplex. Then, neglecting the back reaction Equation 1, the displacement fraction is given as a function of time by where k 1 is the rate constant of strand displacement. Therefore, the time-dependent normalized fluorescence signal plots are fitted into from which the k 1 could be obtained. If the initial concentration of strand I (in this case 100 nm) is in large excess over the SR duplex (in this case 20 nm), then the displacement fraction is given as a function of time by Therefore, the time-dependent normalized fluorescence signal plots are fitted into from which the k 1 could be obtained. S3

4 Text S3: G-quadruplex-Based Toehold Activation Strategy for Controllable DNA Strand Displacement by Sr 2+ Table S1 DNA oligonucleotide sequences used in G-quadruplex systems by Sr 2+. Name Sequence (from 5 to 3 ) a R S G2 I G2 _0 I G2 _6 I G2 _7 I G2 _8 I G2 _TA I G2 _AA S G3 I G3 _0 I G3 _6 I G3 _7 I G3 _8 I G3 _10 S G4 I G4 _0 I G4 _6 I G4 _7 I G4 _8 I G4 _10 S G2 _T1 I G2 _T1 S G2 _T3 I G2 _T3 S G2 _T4 TET-A b ACTAATCCTCAGATCCAGCTAGTGTC-TAMRA GACGCACGACTGGTTGGTT c -GACACTAGCTGGATCTGAGGATTAGTA b ACTAATCCTCAGATCCAGCTAGTGTC-A c A d TGGTTGGTA d ACTAATCCTCAGATCCAGCTAGTGTC-AATGGTTGGTAGTCGTG ACTAATCCTCAGATCCAGCTAGTGTC-AATGGTTGGTAGTCGTGC ACTAATCCTCAGATCCAGCTAGTGTC-AATGGTTGGTAGTCGTGCG ACTAATCCTCAGATCCAGCTAGTGTC-AATGGTAGGTA GTCGTGC ACTAATCCTCAGATCCAGCTAGTGTC-AATGGAAGGTA GTCGTGC GACGCACGACTGGGTTAGGGTT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGTTAGGGTA ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGTTAGGGTAGTCGTG ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGTTAGGGTAGTCGTGC ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGTTAGGGTAGTCGTGCG ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGTTAGGGTAGTCGTGCGTC GACGCACGACTGGGGTTTTGGGGTT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGGTTTTGGGGTA ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGGTTTTGGGGTAGTCGTG ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGGTTTTGGGGTAGTCGTGC ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGGTTTTGGGGTAGTCGTGCG ACTAATCCTCAGATCCAGCTAGTGTC-AATGGGGTTTTGGGGTAGTCGTGCGTC GACGCACGACTGGTGGTT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-AATGGTGGTAGTCGTGC GACGCACGACTGGTTTGGTT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-AATGGTTTGGTAGTCGTGC GACGCACGACTGGTTTTGGTT-GACACTAGCTGGATCTGAGGATTAGTA I G2 _T4 ACTAATCCTCAGATCCAGCTAGTGTC-AATGGTTTTGGTAGTCGTGC a The G-rich segments are underlined and the CS sequences are italicized. b The mismatched da nucleotides are designed to ensure the free state of the fluorophore TET. c The complementary pair dt-da is designed to enhance the stability of tetraplexes. d The da nucleotide is designed to form a bulge structure at the end of a chair type G-quadruplex, which is applied to increase the stability of G-quadruplex. S4

5 Figure S1. Designs of invading strand (I G ) with the different length of complementary single-stranded segment (CS). Figure S2. Effect of the concentrations of strand I G on the strand displacement reaction between the S G R duplex and strand I G in the absence of Sr 2+. The S G R duplex (1 ml, 20 nm) was placed in a cuvette, and strand I G (5 µl at the proper concentration) was added and mixed quickly within 30 s. When the concentration of strand I G was increased from 20 nm to 100 nm, the decrease of fluorescence signal in 30 min was from 18% to 55%, which suggesting that the DNA strand displacement between S G R duplex and strand I G in the absence of Sr 2+ is slow. Strand I G and the SR duplex at 1:1 ratio (fluorescence signal was decreased less than 20% in the absence of Sr 2+ ) was chosen in the following experiments to verify if the addition of Sr 2+ can effectively regulate the strand displacement reaction. S5

6 Figure S3. Effect of the concentrations of strand I G on the strand displacement reaction between the S G R duplex and strand I G in the presence of 20 mm Sr 2+. The S G R duplex (1 ml, 20 nm) with 20 mm Sr 2+ was placed in a cuvette, and strand I G (5 µl at the proper concentration) was added and mixed quickly within 30 s. Figure S4. Effect of the different ions (Sr 2+, K +, Na +, NH + 4, and Mg 2+ ) on the strand displacement rate. The reaction mixture contained 20 nm S G R duplex, 20 nm strand I G, and 10 mm ion. The fluorescence signals were monitored at 30 min. The error bars represent the standard deviation of three measurements. S6

7 Text S4: G-quarduplex-Based Toehold Activation Strategy for Controllable DNA Strand Displacement Reaction by ATP Table S2 DNA oligonucleotide sequences used in G-quadruplex systems by ATP. Name Sequence (from 5 to 3 ) a R S A I A _2 I A _3 I A _4 I A _5 I A _6 TET-AACTAATCCTCAGATCCAGCTAGTGTC-TAMRA CGCTATATACCTGGGGGAGTAT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-AGCGGAGGAAGGTAT ACTAATCCTCAGATCCAGCTAGTGTC-AGCGGAGGAAGGTATA ACTAATCCTCAGATCCAGCTAGTGTC-AGCGGAGGAAGGTATAT ACTAATCCTCAGATCCAGCTAGTGTC-AGCGGAGGAAGGTATATA ACTAATCCTCAGATCCAGCTAGTGTC-AGCGGAGGAAGGTATATAG a The G-rich segments are underlined and the CS sequences are italicized. Figure S5. Principle of the G-quadruplex-based toehold activation strategy by ATP. The anti-atp aptamer was split into two fragments: one fragment was designed in the toehold and the other was included in strand I A. In the presence of ATP, the toehold and strand I A would assemble to form intact structures by binding ATP that promotes the following strand displacement. Displacement reaction is monitored by quenching of fluorescence from strand R. S7

8 Figure S6. Dynamic control of strand displacement kinetics. 20 nm strand IA and 2 mm ATP were added successively into 20 nm SAR duplex. Figure S7. Native PAGE (12%) analysis. Lane 1: 2 µm strand SA, lane 2: 2 µm strand IA, lane 3: 2 µm SAR duplex, lane 4: 2 µm SAIA duplex, lane 5: 2 µm SAR duplex and 2 mm ATP, lane 6: 2 µm SAR duplex and 2 µm strand IA, lane 7: 2 µm SAR duplex, 2 µm strand IA and 2 mm ATP. All of the samples were incubated at room temperature for 30 min. S8

9 Figure S8. CD spectroscopy analysis for the formation of G-quadruplex. 0.5 µm S A R duplex was mixed with either 0.5 µm strand I A, 2 mm ATP, or both, and incubated at room temperature for 30 min. Figure S9. Effect of the concentrations of strand I A on the strand displacement reaction between the S A R duplex and strand I A in the absence of ATP. The S A R duplex (1 ml, 20 nm) was placed in a cuvette, and strand I A (5 µl at the proper concentration) was added and mixed quickly within 30 s. When the concentration of strand I A was increased from 20 nm to 1000 nm, the decrease of fluorescence signal in 30 min was from 5% to 42%, which suggesting that the DNA strand displacement between the S A R duplex and strand I A in the absence of ATP is slow. Strand I A and the S A R duplex at 5:1 ratio (fluorescence signal was decreased less than 10% in the absence of ATP) was chosen in the following experiments to verify if the addition of ATP can effectively regulate the strand displacement reaction. S9

10 Figure S10. Effect of the CS length on the fluorescence discrimination at 30 min in the absence or presence of ATP. Initial concentrations: 20 nm S A R duplex, 100 nm strand I A, and 2 mm ATP. The error bars represent the standard deviation of three measurements. The 4 nt CS length was chosen for further experiments, because the fluorescence signal can be regulated over the widest range (60%) for 4 nt CS length toehold binding. Figure S11. Effect of the concentrations of ATP on the strand displacement kinetics. In a typical experiment, the S A R duplex (1 ml, 20 nm) was placed in a cuvette, and then both strand I A (5 µl, 20 µm) and ATP (2 µl at the proper concentration) were added and mixed quickly within 30 s to initiate the reaction. S10

11 Figure S12. Effect of ATP, TTP, CTP, and GTP on the strand displacement rate. The reaction mixture contained 20 nm S A R duplex, 100 nm strand I A and 0.2 mm nucleoside triphosphate (ATP, TTP, CTP, or GTP). The fluorescence signals were monitored at 30 min. The error bars represent the standard deviation of three measurements. Text S5: i-motif-based Toehold Activation Strategy for Controllable DNA Strand Displacement Table S3 DNA oligonucleotide sequences used in i-motif systems. Name Sequence (from 5 to 3 ) a R S C2 I C2 _0 I C2 _10 I C2 _11 S C3 I C3 _0 I C3 _10 I C3 _11 S C4 I C4 _0 I C4 _10 I C4 _11 I C4 _12 TET-AACTAATCCTCAGATCCAGCTAGTGTC-TAMRA TCTCCAATTCTACCTAACCT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-ACCTAACC ACTAATCCTCAGATCCAGCTAGTGTC-ACCTAACCTAGAATTGGA ACTAATCCTCAGATCCAGCTAGTGTC-ACCTAACCTAGAATTGGAG TCTCCAATTCTACCCTAACCCT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-ACCCTAACCC ACTAATCCTCAGATCCAGCTAGTGTC-ACCCTAACCCTAGAATTGGA ACTAATCCTCAGATCCAGCTAGTGTC-ACCCTAACCCTAGAATTGGAG TCTCCAATTCTACCCCTAACCCCT-GACACTAGCTGGATCTGAGGATTAGTA ACTAATCCTCAGATCCAGCTAGTGTC-ACCCCTAACCCC ACTAATCCTCAGATCCAGCTAGTGTC-ACCCCTAACCCCTAGAATTGGA ACTAATCCTCAGATCCAGCTAGTGTC-ACCCCTAACCCCTAGAATTGGAG ACTAATCCTCAGATCCAGCTAGTGTC-ACCCCTAACCCCTAGAATTGGAGA a The C-rich segments are underlined and the CS sequences are italicized. S11

12 Figure S13. Native PAGE (12%) analysis. Lane 1: 2 µm strand S C at ph 7.5, lane 2: 2 µm strand I C at ph 7.5, lane 3: 2 µm S C R duplex at ph 7.5, lane 4: 2 µm S C I C duplex at ph 7.5, lane 5: 2 µm S C R duplex at ph 5.5, lane 6: 2 µm S C R duplex and 2 µm strand I C at ph 7.5, lane 7: 2 µm S C R duplex and 2 µm strand I C at ph 5.5. All of the samples were incubated at room temperature for 30 min. Figure S14. CD spectroscopy analysis for the formation of the i-motif. 0.5 µm S C R duplex was mixed with or without 0.5 µm strand I C in different ph solutions, and incubated at room temperature for 30 min. S12

13 Figure S15. Effect of the number of C C + pairs and the CS lengths on the strand displacement rate. Three C-rich segments, (C 2 TA 2 C 2 ), (C 3 TA 2 C 3 ), and (C 4 TA 2 C 4 ), which can form stable intermolecular i-motifs in slightly acidic solutions, 5 were used in the design of toeholds. The formed i-motifs composed of four, six, and eight C C + pairs are denoted as C2, C3, and C4, respectively. Initial concentrations: 20 nm S C R duplex and 100 nm strand I C at ph 5.4. The error bars represent the standard deviation of three measurements. Figure S16. Effect of the concentrations of strand I C on the strand displacement reaction between the S C R duplex and strand I C at ph 7.5. The S C R duplex (1 ml, 20 nm) was placed in a cuvette, and strand I C (5 µl at the proper concentration) was added and mixed quickly within 30 s. When the concentration of strand I C was increased from 20 nm to 1000 nm, the decrease of fluorescence signal in 30 min was from 12% to 27%, which suggesting that the DNA strand displacement between the S C R duplex and strand I C at ph 7.5 is slow. Strand I C and the S C R duplex at 5:1 ratio (fluorescence signal was decreased less than 20% at ph 7.5) was chosen for further experiments to verify if the solution acidity can effectively regulate the strand displacement reaction. S13

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