Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2019 Supporting Information: Aptamer-based regulation on transcription circuits Linqiang Pana*, Yingxin Hu †a,b Taoli Dingc, Chun Xie a, Zhiyu Wanga, Zhekun Chena, Jing Yangd*, Cheng Zhange*, a Key Laboratory of Image Information Processing and Intelligent Control of Education Ministry of China, School of Automation, Huazhong University of Science and Technology, Wuhan, 430074, Hubei, China b College of Information Science and Technology, Shijiazhuang Tiedao University, Shijiazhuang, 050043, Hebei, China c Department of Biomedical Engineering, College of engineering, Peking University, Beijing 100871, China d School of Control and Computer Engineering, North China Electric Power University, Beijing 102206, China. e Institute of Software, School of Electronics Engineering and Computer Science, Peking University, Beijing, 100871, China † The author contributes equally to the first author. 1 Table of Contents S1: MATERIAL AND METHODS ...................................................................................................... 3 S2: Experimental details .................................................................................................................... 4 S3: The design of reporter probe ...................................................................................................... 5 S4: Taq polymerase-assisted activator circuit ................................................................................ 6 S4.1: Effect of the buffer in the reaction ........................................................................................ 7 S4.2: Effect of the concentration of DNA polymerase in the reaction .......................................... 8 S4.3: Effect of the concentration of T7 RNA polymerase in the reaction ..................................... 8 S4.4: Effect of the concentration of DNA template in the reaction ............................................... 9 S5: Suppression of transcription using aptamer repressor ........................................................ 10 S6: DNA activator circuit .................................................................................................................. 14 S7: Two-level cascading transcription circuits .............................................................................. 14 S8: Regulation of transcription circuit by enzyme regulators ..................................................... 15 S9: The Simulation models .............................................................................................................. 17 S9.1: The Simulation model of polymerase-triggered transcription circuit................................. 17 S9.2: The Simulation model of two-level cascading transcription circuit ................................... 18 S9.3 Python code for calculating reaction rate constant .............................................................. 19 S10: DNA Sequences ....................................................................................................................... 21 2 S1: MATERIAL AND METHODS Materials All DNA strands were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Unmodified DNA strands were purified by polyacrylamide gel electrophoresis (PAGE), and modified DNA strands with fluorophore and quencher were purified by high-performance liquid chromatography (HPLC). The DNA oligonucleotides were dissolved in water as the stock solution and quantified using Nanodrop 2000, and absorption intensities were recorded at λ = 260 nm. The sequences of all oligonucleotides are listed in Table S1. Thermus aquaticus DNA polymerase (Taq DNA polymerase) was purchased from Takara (Takara Biomedical Technology (Beijing) Co., Ltd.). M.SssI, S-adenosylmethionine (SAM), HpaII, T7 RNA polymerase, dNTP, and rNTP were purchased from New England Biolabs. All other chemicals were of analytical grade and used without further purification. DNA hybridization reaction The DNA complexes were formed by mixing corresponding single strands with equal concentrations in 0.6× RNA Pol reaction buffer. The mixture was annealed in a polymerase chain reaction (PCR) thermal cycler at the reaction condition of 85°C for 5 min, 65 °C for 30 min, 50 °C for 30min, 37 °C for 30min, 25 °C for 30min, and finally kept at 25 °C. DNA polymerase primer extension reaction Taq DNA polymerase (10U/mL or 25U/mL) was mixed with dNTPs (0.25 mM) and DNA template (10nM or 20nM) in 0.6×RNA Pol reaction buffer and then incubated at 25 °C for 90- 160 minutes. RNA polymerase transcription reaction T7 RNA Polymerase (2U/uL) was mixed with rNTPs (1.5 mM), DNA template (10nM or 20nM) in 0.6×RNA Pol reaction buffer to transcribe RNA at 25°C for 90-160 minutes. Fluorescent experiments All experiments were performed in 0.6×RNA Pol reaction buffer using real-time fluorescence PCR (Agilent Technologies). In a typical reaction, the total volume of the solution was 25μL for detection. The FAM fluorescence was monitored at 4 min intervals. Here, fluorescence data were processed to make the initial fluorescent signal value correspond to zero. The fluorescence results were obtained by the average values from at least three times repeat experimental results. Unless specifically mentioned, all the experiments were conducted at 25°C. PAGE experiments The samples were mixed with 6× loading buffer (Takara) or 36% glycerin solution and 3 subjected to electrophoresis analysis on a 12% polyacrylamide gel. The analysis was carried out in 1×TAE buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, pH 8.0) supplemented with 12.5 mM MgCl2 at 90 V for 1-2 hours at 4°C. After stains all (Sigma-Aldrich) staining, Gels were imaged using the scanner. S2: Experimental details (1) Experimental details of the Taq polymerase-assisted transcription: (T1/I1) was mixed with Taq polymerase, T7 RNA polymerase, dNTP, rNTP and (F/Q). The solution was subjected to fluorescence measurement after mixing. The sample was subjected to electrophoresis analysis after incubation at 25°C for 3 hours. For gel analysis, [T1/I1] =20nM, [Taq] = 25U/mL, [F/Q] = 800nM. For fluorescence assay, [T1/I1] = 10nM, [Taq] = 25U/mL, [F/Q] = 400nM. (2) Experimental details of the aptamer-inhibited transcription: the different concentrations of A/B were mixed with DNA polymerase, dNTP and incubated for 20 minutes, followed by the addition of the mixture including T7 RNA polymerase, rNTP, (T1/I1) and (F/Q). The solution was subjected to fluorescence measurement after mixing. The sample was subjected to electrophoresis analysis after incubation at 25°C for 2 hours. For gel analysis, [T1/I1] = 20nM, [Taq] = 25U/mL, [F/Q] = 800nM. For fluorescence assay, [T1/I1] = 10nM, [F/Q] = 400nM. (3) Experimental details in Figure 2, (A/B) was mixed with different concentrations of ‘B*’, DNA polymerase, dNTP and incubated for 20 minutes, followed by the addition of the mixture including T7 RNA polymerase, rNTP, the annealed (T1/I1) and (F/Q). The solution was subjected to fluorescence measurement after mixing. The sample was subjected to electrophoresis analysis after incubation at 25°C for 2 hours. For gel analysis, [T1/I1] = 20nM, [Taq] = 25U/mL, [A/B] = 50nM, [F/Q] = 800nM. For fluorescence assay, [T1/I1] = 10nM, [Taq] = 10U/mL, [F/Q] = 400nM, [A/B] = 30nM. (4) Experimental details in two-level cascading circuits (Figure 3): (A/B) was mixed with Taq polymerase, dNTP and incubated for 20 minutes. Then the incubated sample was added into the mixture in the presence or in the absence of upstream input ‘I2’ containing T7 RNA 4 polymerase, rNTP, (T2/D2), (T1/I1) and the reporter probe (F/Q). The solution was subjected to fluorescence measurement after mixing. The sample was subjected to electrophoresis analysis after incubation at 25°C for 3 hours. For gel analysis, [A/B] = 50nM, [Taq] = 25U/mL, [T1/I1] = 20nM, [F/Q] = 800nM. For fluorescence assay, [A/B] =50nM, [Taq] = 25U/mL, [T1/I1] = 10nM, [F/Q] = 400nM. In Figure 5e, [T2/D2] =0,1,2,5, and 10 nM, [I2] = 0,1,2,5, and 10nM. (5) Experimental details in the enzyme-controlled switch circuit in Figure 4, (A-msi/B-msi) complex incubated with HpaII (in the presence or absence of M.SssI) for 2 hours, the digested product were heated at 80°C for 20 minutes to deactivate the enzyme, followed by the addition of a mixture that contained DNA polymerase, dNTP and incubated for 20 minutes. Then the incubated sample was added into the mixture including T7 RNA polymerase, rNTP, (T1/I1) and reporter probe (F/Q). The solution was subjected to fluorescence measurement after mixing. The sample was subjected to electrophoresis analysis after incubation for 2 hours. For gel analysis, [M.SssI] = 0.5,1,3,4,5,10U/mL, [HpaII] = 10U/mL, [A-msi/B-msi] = 50nM, [Taq] = 25U/mL, [T1/I1] = 20nM, [F/Q] = 800nM. For fluorescence assay, [M.SssI] = 0.3,0.6,0.9,1.2,1.5,3U/mL, [HpaII] = 6U/mL, [A-msi/B-msi] = 30nM, [Taq] = 10U/mL, [T1/I1] =10nM and [F/Q] = 400nM. S3: The design of the reporter probe The 5’ end of strand ‘F’ is labeled with a FAM dye and the 3’ end of strand ‘Q’ is labeled with a BHQ quencher such that the fluorescence is low when the reporter probe is formed due to FRET while the fluorescence is high when strand ‘Q’ is free. To avoid the undesirable elongation of the upper strand NF in the complex (NF/Q) by DNA polymerase, one base mismatch (G) was added at the 3’ end of the sequence of upper strand named as ‘F’. Because the terminal mismatching stalled action of the polymerase at