Electronic Supplementary Material A green method of staining DNA in polyacrylamide gel electrophoresis based on fluorescent copper nanoclusters synthesized in situ Xiaoli Zhu 1, Hai Shi 1, Yalan Shen 1, Bin Zhang 1, Jing Zhao 1, and Genxi Li 1,2 ( ) 1 Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China 2 State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, China Supporting information to DOI 10.1007/s12274-015-0778-y Experimental details Chemicals and materials. Oligonucleotides (Guaranteed Oligos, HPLC-purified) were synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China). Acrylamide and bis-acrylamide were also purchased from Sangon. Magnesium chloride hexahydrate (MgCl 2 6H 2 O), copper sulfate pentahydrate (CuSO 4 5H 2 O), potassium chloride (KCl), sodium chloride (NaCl), sodium phosphate monobasic dihydrate (NaH 2 PO 4 2H 2 O), disodium hydrogen phosphate dihydrate (Na 2 HPO 4 2H 2 O), 3-(N-morpholino)-propane sulfonic acid (MOPS), tris(hydroxymethyl)amino methane (Tris), and ascorbic acid were purchased from Sigma Aldrich. All the reagents were of analytical reagent grade. All solutions were prepared with doubly distilled water, which was purified with a Milli-Q purification system (Branstead, USA) to a specific resistance of > 18 MΩ cm. Preparation of DNA samples. Double stranded DNA (dsdna) and tweezer-structured DNA were prepared by dissolving single stranded oligonucleotides and their corresponding complementary strands in a MOPS buffer solution (10 mm MOPS, 50 mm MgCl 2, ph 7.5). The solution was then heated to 95 C and slowly cooled down to 25 C in about 2 h to ensure the hybridization proceeded completely. The sample of a hybridization chain reaction (HCR) was prepared by incubating three oligonucleotides (a promoter DNA P1, a probe H1, and a probe H2, sequences shown in the ESM) together in a buffer solution (50 mm Na 2 HPO 4, 0.5 M NaCl, ph 6.8) at 37 C for 2 h. The probes H1 and H2 had been preheated to 95 C and slowly cooled down to 25 C to allow the self-folding into a stem loop structure. During the incubation, the promoter P1 might trigger the alternate hybridization of H1 and H2 to form a long double stranded chain. DNA-templated synthesis of copper nanoclusters. In order to obtain CuNCs with high fluorescence intensity for the staining, we first optimized the synthesis condition in the absence of gel preliminarily. Ascorbic acid and CuSO 4 were added into a DNA solution successively and incubated together for 15 min in dark (dsdna 30/40 was adopted, sequences shown in the ESM, final concentrations of ascorbic acid, CuSO 4, and DNA were 1 mm, 100 μm, and 0.5 μm, respectively). Several variables including ph, buffer, ion strength and temperature were Address correspondence to Genxi Li, email: genxili@nju.edu.cn.
taken into consideration. The produced CuNCs were characterized by UV vis spectrometry, fluorescence spectroscopy, and photographing under UV light or a Gel Doc XR Imaging System (BioRad). Polyacrylamide gel electrophoresis and staining. 12% non-denaturing polyacrylamide gel electrophoresis was carried out in a Tris-boric acid-disodium EDTA (TBE) buffer at 120 V constant voltage for about 1.5 h. After electrophoresis, the polyacrylamide gel was stained by fluorescent copper CuNCs or EtBr as control. In the case of CuNCs staining, the polyacrylamide gel was first immersed in a 50 ml MOPS buffer solution (10 mm MOPS, 50 mm MgCl 2, ph 7.5) containing 100 μm CuSO 4 for 15 min. Then, ascorbic acid with a final concentration of 1 mm was added to the solution and incubated for another 15 min to allow the synthesis of CuNCs. The gel was stained twice by repeating the above procedure again to enhance the fluorescence intensity. For EtBr staining, the gel was submerged in an EtBr solution (300 ml, 0.5 μg/ml) for 15 min at 25 C after running electrophoresis. The gel in either case was finally taken out and imaged by using the Gel Doc XR Imaging System. A filter for the excitation of EtBr (~302 nm) was adopted throughout the experiments (an imaging system with an optimal excitation at 343 nm may be better for the CuNCs-based staining). Other parameters of the imaging system were kept as default values. Skin toxicity test. All animal procedures were performed in accordance with institutional and national guidelines and with approval from the Animal Care Ethics Committee of Shanghai University. Adult male Sprague Dawley (SD) rats weighing (465.7 ± 56.9) g at receipt were purchased from Fudan University and housed in groups of 2 in plastic cages. The rats were randomly assigned as control and test groups. The back skin of the rats was treated with depilatory cream and shaved with an electric razor to get a glabrous area of about 20 cm 2. 200 μl of copper sulfate (100 μm) or ascorbic acid (1 mm) or the synthesizing/synthesized CuNCs or double distilled water as control was spread evenly onto the exposed skin area. After 24 h, the treated skin area was washed to remove the reagents; and fresh samples were applied again. The procedure of the treatment was repeated during a month. The local conditions of the treated skin area and the overall state of the rats were observed and recorded continuously by the experimenters. Finally, the rats were put to death. The internal organs were observed; and the liver was weighed to investigate if there was hepatomegaly. Table S1 DNA sequences Name poly(a) 40 poly(t) 40 poly(taa) 40 poly(att) 40 poly(c) 40 poly(cgg) 40 poly(gcc) 40 dsdna 21 dsdna 20/30 dsdna 30/40 HIV 42 Sequence 5'-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA A-3' 5'-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T-3' 5'-TAA TAA TAA TAA TAA TAA TAA TAA TAA TAA TAA TAA TAA T-3' 5'-ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT A-3' 5'-CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC CCC C-3' 5'-CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG CGG C-3' 5'-GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC GCC G-3' 5'-TAC TCA TAC GCT CAT GAC TTC-3' 3'-ATG AGT ATG CGA GTA CTG AAG-5' 3'-GTA TGC AAG TAG TGC TGA TG-5' 5'- CTC ATA CGC TCA TAC GTT CAT CAC GAC TAC-3' 5'- CTC ATA CGC TCA TAC GTT CAT CAC GAC TAC-3' 3'-TCT TAC TGA TGA GTA TGC GAG TAT GCA AGT AGT GCT GAT G-5' 5'-ACT GCT AGA GAT TTT CCA CAC TGA CTA AAA GGG TCT GAG GGA-3' 3'- TGA CGA TCT CTA AAA GGT GTG ACT GAT TTT CCC AGA CTC CCT-5' www.editorialmanager.com/nare/default.asp
Name Hly 44 HBV 30 Tweezer-structured DNA Hairpin-structured DNA HCR amplification DNA RNA hybridization Sequence 5'-TCT CCG CCT GCA AGT CCT AAG ACG CCA ATC GAA AAG AAA CAC GC-3' (Continued) 3'-AGA GGC GGA CGT TCA GGA TTC TGC GGT TAG CTT TTC TTT GTG CG-5' 5'-ATA CCA CAT CAT CCA TAT AAC TGA AAG CCA-3' 3'-TAT GGT GTA GTA GGT ATA TTG ACT TTC GGT-5' 5'-TAA TTA TTT ATT ATA TAT ACC CCC CCA TAT ATA TTA TTT ATT AAT-3' 5'-TTA TAT GAA ACC AGA AAA TAT ATA TAA TAA ATA ATT A-3' 5'-ATT AAT AAA TAA TAT ATA TAA AAC CAA GGA ATT ATA T-3' 5'-ACC TCA TTG TAT AGC TGA GGT AGT AGG TTG TAC AAC TAT ACA ACC TAC TAC CT-3' P1: 5'-AGT CTA GGA TTC GGC GTG GGT TAA-3' H1: 5'-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3' H2: 5'-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3' RNA: 5'-GGG CGA CCC UGA UGA GGC CUU CGG GCC GAA ACG GUG AAA GCC GUC GGU CGC CC-3' DNA: 3'-CCC GCT GGG ACT ACT CCG GAA GCC CGG CTT TGC CAC TTT CGG CAG CCA GCG GG-5' Figure S1 Effect of ph on the formation of DNA-templated fluorescent CuNCs in a buffer solution (10 mm MOPS containing 150 mm NaCl, 0.5 μm dsdna 30/40, 100 μm CuSO 4, and 1 mm ascorbic acid). Photographs were taken 15 min after mixing all the reagents together. Figure S2 Formation of DNA-templated fluorescent CuNCs in different buffer solutions with a ph of 7.5. The concentrations of MOPS, Tris, and PBS are all 10 mm. Different ions are also involved, the kinds and concentrations of which have been marked on the top of the figure. Ref. [1] represents the buffer condition from Ref. [1]: 10 mm MOPS buffer containing 150 mm NaCl. Other conditions are the same as that in Fig. S1. www.thenanoresearch.com www.springer.com/journal/12274 Nano Research
Figure S3 Effect of ionic strength on the formation of DNA-templated fluorescent CuNCs in 10 mm MOPS buffer solution (ph 7.5). The concentration of NaCl from 1 to 8 is 25, 50, 75, 100, 200, 300, 400, and 500 mm, respectively; MgCl 2 : 0, 5, 15, 25, 50, 100, 150, 200 mm; KCl: 0, 25, 50, 100, 200, 300, 400, 500 mm. C represents a control group, in which DNA is absent. The concentrations of the ions of the control group are 100 mm NaCl, 50 mm MgCl 2, and 100 mm KCl, respectively. Other conditions are the same as that in Fig. S1. Figure S4 Effect of temperature on the formation of DNA-templated fluorescent CuNCs in 10 mm MOPS buffer solution containing 50 mm MgCl 2 (ph 7.5). Other conditions are the same as that in Fig. S1. Figure S5 Fluorescence of the DNA-templated fluorescent CuNCs under UV light. The CuNCs were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl 2, 0.5 μm dsdna 30/40, 100 μm CuSO 4, and 1mM ascorbic acid) at 25 C for different reaction time. Figure S6 Fluorescence spectra of the DNA-templated fluorescent CuNCs. Other conditions are the same as that in Fig. S5. www.editorialmanager.com/nare/default.asp
Figure S7 UV vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl 2, 100 μm CuSO 4, and 1 mm ascorbic acid) at 25 C in the absence of DNA template for different reaction time. Figure S8 UV vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl 2, 100 μm CuSO 4, and 1 mm ascorbic acid) at 25 C in the presence of ssdna 30 (0.5 μm) as the template for different reaction time. The absorption of ascorbic acid and DNA overlaps at 260 nm. The real absorption of DNA emerges when the ascorbic acid is exhausted. Figure S9 UV vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl 2, 100 μm CuSO 4, and 1 mm ascorbic acid) at 25 C in the presence of dsdna 30/40 (0.5 μm) as the template for different reaction time. The absorption of ascorbic acid and DNA overlaps at 260 nm. The real absorption of DNA emerges when the ascorbic acid is exhausted. www.thenanoresearch.com www.springer.com/journal/12274 Nano Research
Figure S10 UV vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl 2, 100 μm CuSO 4, and 1 mm ascorbic acid) at 25 C in the presence of dsdna 20/30 (0.5 μm) as the template for different reaction time. The absorption of ascorbic acid and DNA overlaps at 260 nm. The real absorption of DNA emerges when the ascorbic acid is exhausted. Figure S11 The ladder of DNA marker II (0.5 μg) in polyacrylamide gel by using the CuNCs-based staining under different conditions. The leftmost band is performed under the optimized condition (10 mm MOPS, ph 7.5, 50 mm MgCl 2, at 25 C). Others are different from the optimized condition with single-variable, which has been marked over the ladder. Figure S12 The ladder of DNA marker II with different molar quantities in polyacrylamide gel by using EtBr staining. www.editorialmanager.com/nare/default.asp
Figure S13 The fluorescence of different samples stained by CuNCs (left) and EtBr (right) in polyacrylamide gel. From lane 1 to 9: poly(a) 40, poly(t) 40, poly(taa) 40, poly(att) 40, poly(c) 40, poly(cgg) 40, dsdna (A-T)40, dsdna (TAA-ATT)40, dsdna (CGG-GCC)40. The subscript number indicates the number of bases/base pairs. The concentration of all nucleic acids is fixed at 0.5 μm. The bands in red box are those that can be stained by both CuNCs and EtBr, while green and blue boxes show the bands that can only be stained by CuNCs or EtBr. Figure S14 The skin toxicity of CuSO 4, ascorbic acid, and synthesizing CuNCs on adult male SD rats. The overall and local states of the rats after treated with 100 μm CuSO 4, or 1 mm ascorbic acid, or synthesizing CuNCs (CuSO 4 and ascorbic acid were spread onto the skin simultaneously) for 1 d, 7 d, and 30 d are presented. The corresponding weights of the rats as well as the liver weights after dissection are shown in the table beneath the photos. Experiments on two other groups working as duplicate samples were also conducted without shown. Reference [S1] Rotaru, A.; Dutta, S.; Jentzch, E.; Gothelf, K.; Mokhir, A. Selective dsdna-templated formation of copper nanoparticles in solution. Angew. Chem. Int. Ed. 2010, 49, 5665 5667. www.thenanoresearch.com www.springer.com/journal/12274 Nano Research