Supporting Information. Universal Strategy To Engineer Catalytic DNA Hairpin Assemblies for Protein Analysis

Transcription

1 Supporting Information Universal Strategy To Engineer Catalytic DNA Hairpin Assemblies for Protein Analysis Yanan Tang, Yanwen Lin, Xiaolong Yang, Zhixin Wang, X. Chris Le, and Feng Li, * Department of Chemistry and Center for Biotechnology, Brock University, St. Catharines, Ontario, Canada, L2S 3A1 Department of Chemistry and Department of Laboratory Medicine and Pathology, University of Alberta, Alberta, Canada, T6G 2G3 *Corresponding author. fli@brocku.ca

2 Materials and Reagents Human α-thrombin, human prostate specific antigen (PSA), streptavidin from Streptomyces avidinii, biotin, bovine serum albumin (BSA), human serum, streptavidin coated magnetic microparticles, ROX reference dye, and magnesium chloride hexahydrate (MgCl 2 6H 2 O) were purchased from Sigma (Oakville, ON, Canada). Recombinant human epidermal growth factor receptor 2 (HER2), biotinylated anti-her2 polyclonal antibody (goat IgG), biotinylated anti-psa polyclonal antibody, fluorescein labeled streptavidin, and biotinylated normal goat IgG control were purchased from R&D Systems (Minneapolis, MN). Phosphate buffer saline (PBS), Tween 20, diamidino-2- phenylindole (DAPI) solution (1 mg/ml), McCoy s 5A modified medium, penicillin/streptomycin (100 ), and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA). NANOpure H 2 O (> 18.0 MΩ), purified using an Ultrapure Milli-Q water system, was used for all experiments. All DNA samples were purchased from Integrated DNA Technologies (Coralville, IA) and purified by high performance liquid chromatography (HPLC). The DNA sequences and modifications are listed in Table S1. Prepare DNA probes for protein-responsive catalytic hairpin assembly (CHA) To prepare DNA probes for the detection of thrombin, two distinct thrombin aptamers were directly incorporated to the end of TB and B*C during DNA synthesis (Table-1). To prepare DNA probes for PSA or HER2, 25 µl 2.5 µm biotinylated probe TB or B*C was mixed with equal volume of 2.5 µm streptavidin and incubated at 37 C for 30 min. To this reaction mixture, 50 µl 1.25 µm biotinylated PSA or HER2 polyclonal antibodies was then added. The solution was incubated at 25 C for 30 min. The prepared DNA probe was then diluted to 100 nm with a solution containing 1 PBS, 0.01% BSA, and 1 mm biotin. The fluorescent displacement beacon FQ was prepared at a final concentration of 5 µm by mixing 20 µl 50 µm FAM-labeled F with 20 µl 100 µm dark quencher-labeled Q in 160 µl PBS-Mg buffer (1 PBS, 10 mm MgCl 2, 0.05% Tween20). The mixture was heated to 90 C for 5 min and then the solution was allowed to cool down slowly to 25 C in a period of 2 hours. DNA probe F Q that was used to clean up H1 with disfavored secondary structures was prepared using the same protocol

3 as for FQ. A solution of 100 µl 5 µm biotinylated F Q probe was then incubated with 100 µl 10 mg/ml streptavidin coated magnetic beads (1 µm diameter) at room temperature for 1 hour, washed three time using PBS-Mg buffer, and finally re-suspended in 200 µl PBS-Mg buffer. Before each CHA reaction, 100 µl 500 µm H1 was mixed 10 µl F Q -magnetic beads for 30 min to remove disfavored probes. Protein-responsive CHA For a typical protein-responsive CHA reaction, a reaction mixture containing 10 nm TB, 10 nm B*C, 50 nm H1 (purified using F Q -magnetic beads), 50 nm H2, 25 nm FQ, 50 nm ROX reference dye, varying concentrations of the target protein, and PBS-Mg buffer was incubated at 37 C in a 96-well microplate. Fluorescence signals were measured every 5 min from the microplate using a multi-mode microplate reader (SpectraMax i3, Molecular Devices). The excitation/emission for FQ beacon were 485/515 nm and the excitation/emission for the ROX reference dye were 535/595 nm. The measured fluorescence was normalized so that 1 normalized unit (n.u.) of fluorescence corresponded to fluorescent signal generated by 1 nm positive control (P). This normalization was achieved using a positive control containing 25 nm P, 25 nm FQ, 50 nm ROX, and 1% BSA in PBS-Mg buffer, and a negative control containing identical reagents in positive control except that there was no P added. For experiments in Figure 2, Figure 3, Figure S1, and Figure S2, DNA probe T9B7 was used to construct thrombinresponsive CHA. For native polyacrylamide gel electrophoresis characterization, a reaction mixture containing 50 nm TB, 50 nm B*C, 2 µm H1, 1 µm H2, and 25 nm thrombin was incubated at 37 C for 2 hrs. Reaction mixture and various controls were then loaded into 12% PAGE gel and a voltage of 110 V was applied. After electrophoresis, the gel was stained with Ethidium Bromide and imaged using Gel Doc XR+ Imager System (BioRad). The quantitative analysis was carried out using Imaging Lab 5.1 (BioRad). Relative quantifications of all DNA bands were achieved by using 1 µm H2 (Lane 3) as a standard.

4 Cell culture and fixation The human breast adenocarcinoma cell line SK-BR-3, was obtained from ATCC (Manassas, VA) and cultured in McCoy s 5A medium (ATCC) plus 10% FBS and 1% penicillin/streptomycin (100 U/100 µg/ml) at 37 C in a humidified atmosphere of 5% CO 2. For a typical fluorescent imaging experiment, cells were seeded to 96-well plate at a concentration of cells per well. When cultured to 90% confluence, cells were fixed using 4% paraformaldehyde for 30 min and permeabilized at room temperature for 10 min using 0.05% Tween20 in PBS (PBST) buffer. After a brief wash using PBST buffer, cells were stored in PBST buffer containing 1% BSA at 4 C. Cell sensing using HER2-responsive CHA To detect cellular HER2 using CHA, fixed SK-BR-3 cells were mixed with 100 µl reaction solution containing 10 nm HER2-specific TB, 10 nm HER2-specific B*C, 50 nm H1 (pre-purified using magnetic bead), 50 nm H2, 25 nm FQ, 50 nm ROX reference dye, 1% BSA, and PBS-Mg buffer. The reaction mixture was incubated at 37 C and fluorescence increases were measured every 5 min from the microplate using a multimode microplate reader. The excitation/emission for FQ beacon were 485/515 nm and the excitation/emission for the ROX reference dye were 535/595 nm. The measured fluorescence was normalized so that 1 normalized unit (n.u.) of fluorescence corresponded to fluorescent signal generated by 1 nm positive control (P). This normalization was achieved using a positive control containing 25 nm P, 25 nm FQ, 50 nm ROX, and 1% BSA in PBS-Mg buffer, and a negative control containing identical reagents in positive control except that there was no P added. Cell imaging using fluorescent immunostaining and confocal fluorescence microscopy To image cellular HER2 using fluorescent immunostaining, fixed SK-BR-3 cells were blocked with 1% BSA for 30 min and then mixed with biotinylated HER2-specific polyclonal antibodies at a final concentration of 10 nm for 1 hr. 20 nm FITC-labeled streptavidin and 100 nm DAPI were then added and incubated for another 30 min. After incubation, cells were washed for 5 times using PBST buffer to remove unbound

5 antibodies and FITC-labeled streptavidin before fluorescent imaging. Isotype controls were prepared the same way as for the samples, except that nonspecific goat IgG was used instead of HER2-specific antibodies. Fluorescence imaging of fixed cells was performed on an Olympus IX-81 microscope that was coupled with a Yokagawa CSU 1 spinning disk confocal scan-head and Hamamatsu EMCCD cameras with 40 /1.3 Oil and 20 /0.85 Oil objective lenses. A 405 nm pumped diode laser was used for the excitation of nucleus staining dye DAPI. A 491 nm pumped diode laser was used for the excitation of FITC-labeled HER2. The exposure time was set to be 200 ms for DAPI and 600 ms for FITC for both samples and controls.

6 Table S1. DNA sequences and modifications DNA name Sequences TC H1 H2 5 - GCGTGTATC CCATGTGTC CGT GTG ATG-3 5 -CAT CAC ACG GACACATGG GATACACGC CCATCGTGTAC GCGTGTATCC CATGTGTC AAGAGCAC ACTTCTCA GATACACGC GTACACGATGG GCGTGTATC CCATGTGTC CCATCGTGTAC-3 F 5 -FAM-TGAGAAGTGTGCTCTT GACACATG-3 Q 5 - AAGAGCAC ACTTCTCA-Dabcyl-3 P (control) 5 - CATGTGTC AAGAGCAC ACTTCTCA -3 DNA probes for thrombin DNA probes for streptavidin, PSA, and HER2 DNA probes for magnetic separation B*C T9B6 T9B7 T9B8 T9B9 T9B10 TB B*C 5 -GCG TGT ATC CCA TGT GTC-CCT CAC TGA G -TT TTT TTT TT -GGT TGG TGT GGT TGG-3 5 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT T TTT TTT TTT TTT T GTG AGG TT CGT GTG ATG-3 5 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT T TTT TTT TTT TTT AGTG AGG TT CGT GTG ATG-3 5 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT T TTT TTT TTT TA CAGTG AGG TT CGT GTG ATG-3 5 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT T TTT TTT TTT T TCAGTG AGG TT CGT GTG ATG-3 5 -AGT CCG TGG TAG GGC AGG TTG GGG TGA CT T TTT TTT TTT CTCAGTG AGG TT CGT GTG ATG-3 5 -Biotin-TTT TTT TTT TTT TTT T-GTG AGG-TT-CGT GTG ATG-3 5 -GCG TGT ATC CCA TGT GTC-CCT CAC TTT TTT TTT TTT TTT T-Biotin-3 F 5 -Biotin-TGAGAAGTGTGCTCTT GACACATG-3 Q 5 - AAGAGCAC ACTTCTCA-3

7 Table S2. Stability of binding-induced DNA complexes that are characterized through free energies ΔG and melting temperatures T m. DNA Probes Complementary ΔG (kcal/mole) T m ( C) Length (bp) B*C + T9B B*C + T9B B*C + T9B B*C + T9B Note: Free energies and melting temperatures of binding-induced DNA complexes were estimated using OligoAnalyzer (free software from IDT). The ΔG and T m values of B:B* was estimated by considering the binding-induced DNA assembly as a DNA hairpin structure with complementary base pairs ranging from 6 to 9 as the stems and 60 dntp as the loop. This loop length was chosen for estimation because of its similar size to that of the affinity complex.

8 Supporting Figures Figure S1. Specificity of thrombin-responsive CHA. Error bars represent one standard deviation from duplicated analyses.

9 Figure S2. (A) Comparing thrombin-responsive CHA with CHA catalyzed by its DNA counterpart. (B) Amplification factor (AF) is determined as an indicator to characterize the catalytic efficiency of 2 nm thrombin and its DNA counterpart (2 nm). AF is defined as a ratio between fluorescence increase of CHA and that of the same CHA reaction but is arrested at a target-h1 reaction intermediate (no H2 was added). AF = [H1:H2] / [Intermediate] = (I H1:H2, target I H1:H2, blank ) / (I Intermediate, target I Intermediate, blank ), where I H1:H2, target is denoted as fluorescent intensity of protein-responsive CHA in the presence of the target protein, I H1:H2, blank is denoted as fluorescent intensity of protein-responsive CHA in the absence of the target protein; I Intermediate, target is denoted as fluorescent intensity of the reaction intermediate in the presence of the target protein, and I Intermediate, blank is denoted as fluorescent intensity of the reaction intermediate in the absence of the target protein. (C) Schematic illustration of the generation and detection of the target-h1 reaction intermediate for thrombin-responsive CHA. The reaction intermediate is generated by eliminating H2 probes in thrombin-responsive CHA and is detected using the same FQ displacement beacon. As no H2 is added into the reaction, no signal amplification can be achieved. (D) Fluorescence signal increase from the reaction intermediate as a function of time in the presence (blue curve) or absence (red curve) of target thrombin.

10 Figure S3. (A) Schematic illustrating the principle of streptaividin-responsive CHA; (B) Monitor fluorescence signal increase of streptavidin-responsive CHA in real-time; (C) Amplification factor (AF) of streptavidin-responsive CHA as a function of time. AF at each time point is calculated using the following equation: AF = [H1:H2] / [Intermediate] = (I H1:H2, target I H1:H2, blank ) / (I Intermediate, target I Intermediate, blank ), where I H1:H2, target is denoted as fluorescent intensity of protein-responsive CHA in the presence of the target protein, I H1:H2, blank is denoted as fluorescent intensity of protein-responsive CHA in the absence of the target protein; I Intermediate, target is denoted as fluorescent intensity of the reaction intermediate in the presence of the target protein, and I Intermediate, blank is denoted as fluorescent intensity of the reaction intermediate in the absence of the target protein.

11 Figure S4. Detection of human α-thrombin spiked in 10-time diluted human serum sample. End-point fluorescence was measured after incubating the reaction mixture for 150 min. Error bars represent one standard deviation from duplicated analyses.

12 Figure S5. (A) Immunostaining of fixed SK-BR-3 cells using biotinylated HER2 specific polyclonal antibodies and FITC labeled streptavidin. (B) Immunostaining of fixed SK- BR-3 cells using biotinylated nonspecific goat IgG and FITC labeled streptavidin as an isotype control. Scale bars represent 22 µm.