A green staining method of DNA in polyacrylamide gel electrophoresis based on in situ synthesized fluorescent copper nanoclusters

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1 Nano Research DOI /s y Nano Res 1 A green staining method of DNA in polyacrylamide gel electrophoresis based on in situ synthesized fluorescent copper nanoclusters Xiaoli Zhu 1, Hai Shi 1, Yalan Shen 1, Bin Zhang 1, Jing Zhao 1 and Genxi Li 1,2 ( ) Nano Res., Just Accepted Manuscript DOI /s y on April 8, 2015 Tsinghua University Press 2015 Just Accepted This is a Just Accepted manuscript, which has been examined by the peer-review process and has been accepted for publication. A Just Accepted manuscript is published online shortly after its acceptance, which is prior to technical editing and formatting and author proofing. Tsinghua University Press (TUP) provides Just Accepted as an optional and free service which allows authors to make their results available to the research community as soon as possible after acceptance. After a manuscript has been technically edited and formatted, it will be removed from the Just Accepted Web site and published as an ASAP article. Please note that technical editing may introduce minor changes to the manuscript text and/or graphics which may affect the content, and all legal disclaimers that apply to the journal pertain. In no event shall TUP be held responsible for errors or consequences arising from the use of any information contained in these Just Accepted manuscripts. To cite this manuscript please use its Digital Object Identifier (DOI ), which is identical for all formats of publication.

2 TABLE OF CONTENTS (TOC) A green staining method of DNA in polyacrylamide gel electrophoresis based on in situ synthesized fluorescent copper nanoclusters 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 , P. R. China. 2 State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing , P. R. China. Green staining of DNA in polyacrylamide gel electrophoresis is achieved on the basis of in situ synthesized fluorescent copper nanoclusters. It is a promising alternative to the exiting unsafe staining methods.

3 Nano Research DOI (automatically inserted by the publisher) Research Article A green staining method of DNA in polyacrylamide gel electrophoresis based on in situ synthesized fluorescent copper nanoclusters Xiaoli Zhu 1, Hai Shi 1, Yalan Shen 1, Bin Zhang 1, Jing Zhao 1, and Genxi Li 1,2 ( ) Received: day month year Revised: day month year Accepted: day month year (automatically inserted by the publisher) Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014 KEYWORDS staining, nucleic acids, polyacrylamide gel, copper nanoclusters, electrophoresis ABSTRACT The safety of nucleic acids staining dyes has long been plaguing the researchers. Extensive efforts have been made to search for alternatives to the most popular but toxic staining dye, i.e., ethidium bromide (EtBr). However, no staining method can be guaranteed to be safe enough so far. In this paper, we report a green staining method of DNA in polyacrylamide gel electrophoresis, where in situ synthesis of DNA-templated fluorescent copper nanoclusters (CuNCs) in the gel is achieved to make the DNA bands visible under UV light. Moreover, comprehensive performances of this staining method have been conducted and the experimental results show that it has favorable sensitivity, stability, and usability. Meanwhile, in our animal experiments, the two reagents (copper sulfate and ascorbic acid) as well as the synthesized CuNCs have been proven to be non-toxic in contact with skin. In addition, all reagents adopted in this work are readily available and cost-efficient, and the procedure of this method is rather simple. Therefore, this novel staining method by in situ synthesizing DNA-templated fluorescent CuNCs may have great application prospects in the future. Gel electrophoresis of DNA is a well-known analytical technique used for isolation, purification, and identification of DNA fragments. It has become one of the most important techniques in the modern life science, and has been widely applied in molecular biology [1], genetics [2], microbiology [3], biochemistry [4], and forensics [5]. As DNA molecules are colorless, so their progress through the gel during electrophoresis cannot be followed unless a staining procedure is conducted. In 1972, ethidium bromide (EtBr), the most common stain, was first exploited to work as a tag of DNA in gel electrophoresis [6]. It can intercalate into the major groove of DNA and fluoresces under UV light to make the bands of DNA visible. However, owing to the potential human mutagenicity and complex Address correspondence to Genxi Li, genxili@nju.edu.cn.

4 2 Nano Res. waste disposal issues of EtBr, researchers have been constantly seeking for alternative staining methods. Currently, SYBR dyes represented by SYBR Green I have become the most popular alternatives to EtBr. SYBR Green I, an asymmetrical cyanine dye, is reported to be 25 times more sensitive and possibly safer than EtBr [7]. Nevertheless, the safety of SYBR Green I as well as some other emerging stains is far from guaranteed, because there are also opposite results to show their toxicity, and there is no data addressing their mutagenicity or toxicity in humans [8]. Furthermore, despite the performance advantage of some alternatives for staining purposes, many researchers still prefer EtBr since it is considerably less expensive. Thus, a staining method with favorable safety and cost-efficiency is still badly needed. Recently, metallic nanoclusters (MNCs), a newly developed type of fluorescent nanomaterials, have attracted much attention [9-13]. Biomolecule-templated synthesis of fluorescent MNCs provides a concept to link biomolecules with nano-tags in a natural way. DNA has long been recognized as a good template in the synthesis of nanomaterials [14-17]. DNA-templated synthesis of fluorescent copper nanoclusters (CuNCs), silver nanoclusters (AgNCs), and gold nanoclusters (AuNCs) has also been reported successively in recent years [18-20]. Taking CuNCs for example, it has been reported that DNA attached to a silicon surface can be metallized with copper [21]. On this basis, the group of Mokhir found that double stranded DNA (dsdna) could work as a template for the synthesis of fluorescent CuNCs in solution [18]. Since then, thymine-rich single-stranded DNA (ssdna) was also exploited to work a template [22, 23]; and the application of the DNA-templated fluorescent CuNCs in biosensing has been achieved [24-26]. Here, inspired by the need of staining method in electrophoresis and the progress in DNA-templated synthesis of MNCs, we develop a novel staining method of DNA in polyacrylamide gel electrophoresis by in situ synthesizing fluorescent CuNCs in the gel. A simple experimental protocol for the staining is established after extensive trials. The agents involved are common and cheap, and have been proven to be non-toxic in contact with skin. Thus, this staining method provides favorable safety and cost-efficiency, and may have potential applications after further optimization. The experimental conditions for the synthesis of DNA-templated fluorescent CuNCs are first optimized. Since the fluorescence intensity is quite important for staining, it works as the main criterion for judging whether the conditions are suitable. Also because there are so many factors that may affect the fluorescence intensity of the CuNCs-based staining, and the optimization in gel (including the preparation of gel, electrophoresis, and staining) is a tedious work with quite low efficiency, some possible common experimental conditions were firstly optimized in solution to facilitate the process. A dsdna (dsdna30/40, sequences shown in the Electronic Supplementary Material (ESM)), which has been proven to be able to work as the template for the synthesis of fluorescent CuNCs, is adopted here for the condition optimization. Several elements including ph, buffer solution, ion strength, and temperature are taken into account. From the experimental results, the most intense fluorescence is obtained in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl2) at 25 C (Fig. S1~S4 in the ESM). Under this condition, the synthesized CuNCs fluoresce immediately after the mixing of the reactants (CuSO4 and ascorbic acid) with the dsdna. The fluorescence intensity reaches a plateau after about 10 min. The peaks of the excitation and emission are at 343 nm and 584 nm respectively, representing an orange-red color in appearance under UV light (Fig. S5 and Fig. S6 in the ESM). UV-vis spectrometric results show that the period of the formation of CuNCs (including both on DNA template and without template) lasts for 80 to 160 min (a time required to allow the absorption peaks of CuNCs to reach a plateau) (Fig. S7~S10 in the ESM). Because only DNA-templated CuNCs fluoresce and the fluorescence intensity can reach a plateau in only about 10 min, it is reasonable that the CuNCs have priority to form on DNA and afterward form slowly without template. Gel staining result has partially confirmed that the optimized conditions for solution can be also applied to gel (Fig. S11 in the ESM). Thus, those common experimental conditions, i.e., ph, buffer solution, ion strength, and temperature, are adopted without further optimization. Nevertheless, lots of factors e.g.

5 3 the diffusion of copper ions and ascorbic acid in gel, the restricted state of DNA in gel, and the porous morphology of gel should be taken into account. Thus, extensive trials on staining time, sequence of adding reactants, number of times for staining, and etc, have been carried out (Fig. 1, some less important Figure 1 The bands of DNA marker II (0.5 μg) in polyacrylamide gel were stained by CuNCs (only 25, 50, 75, 100 bp are shown on the left of Fig. 1 to avoid being over crowded). The staining time, sequence of adding reactants, and number of times for staining were variable to obtain an optimized condition. The columns A, B, and C correspond to adding of CuSO4 together with, before (15 min earlier), and after (15 min later) the addition of ascorbic acid to the gel-immersed staining solution, respectively. The columns 1 and 2 indicate the number of times for staining, i.e., staining for once and twice. The numbers in the left show the time of the reaction of CuSO4 with ascorbic acid in one staining cycle. The red box indicates the bands under the optimized condition. In comparison, the bands using EtBr staining is also presents. results are not shown). DNA marker II with different length of DNA fragments from 25 bp to 700 bp is adopted as a model to verify the performance of the CuNCs staining (only 25, 50, 75, 100 bp are shown on the left of Fig. 1 to avoid being over crowded). It is interesting that if the gel after electrophoresis is immersed in the reaction system for a certain period and then taken out immediately for imaging, the brightness of the bands is weak. In contrast, if the gel is stained twice, i.e., immersed in two equal but independent reaction systems successively for a certain period, the bands become clear. Further repetition of staining has little effect to the improvement of the brightness or the definition, so staining for twice is set as a standard process for the following experiments. Results also indicate that the performance of CuNCs staining is related to the staining time and the sequence of adding reactants. With all factors considered, the optimized condition of the corresponding gel pattern result, which is marked in the red box (Fig. 1), is finally adopted. Under this optimized condition, all bands are clearly visible, which may rival the performance of EtBr staining. Detailed processes have been shown in the experimental sections and the caption of Fig. 1. Comprehensive performance including sensitivity, stability, and usability of the CuNCs staining is then studied. As is shown in Fig. 2, different amounts of the DNA marker II are loaded, electrophoresed, and stained to investigate the sensitivity. It is observed that the brightness of the DNA bands fades with the decrease of the amount of the DNA marker. All bands except the 25 bp fragment are clearly visible until the molar quantity of the DNA marker decreases to 0.02 μg. Similar sensitivity is obtained while EtBr staining is adopted instead (Fig. S12 in the ESM). If the 25 bp fragment is taken into consideration, the sensitivity of CuNCs staining declines to about 0.3 μg. It has been reported that the fluorescence of CuNCs depends on the sequence and length of the DNA template [18, 22, 23], so here the short DNA fragment may be not as good as a longer one to work as the template. Nevertheless, the performance of CuNCs staining on sensitivity is Nano Research

6 4 Nano Res. acceptable. We next investigate the stability of the CuNCs Figure 2 The ladder of DNA marker II with different molar quantities in polyacrylamide gel by using the CuNCs-based staining under the optimized condition. Figure 3 The gel was placed in (A) a dark and argon environment, (B) a dark and atmospheric environment, (C) a UV light and argon environment, (D) a UV light and atmospheric environment, (E) a dark and nitrogen environment, (F) dark and ascorbic acid (AA) solution (2 mm), for different time before photographing under UV light. The numbers above each photo show the time (min). staining. In experiments, the CuNCs stained gel is placed in different environments for a certain period before photographing under UV light to study the time-, aero-, and photo-stability, respectively. As is shown in Fig. 3A, the fluorescence of the CuNCs-stained bands hardly changes under the dark and argon environment, suggesting that the DNAtemplated CuNCs are stable somehow and there is no self-decay. However, it is noticed that if the gel is exposed to the air (Fig. 3B), the fluorescence disappears gradually in 15 min. This result is reasonable, because copper especially in nanoscale is easy to be oxidized by oxygen [27]. While exposing the gel to UV light for a certain period (Fig. 3C), we observe that there is also a little decay of the fluorescence. Nevertheless, the process is much slower in comparison with the effect of air. This phenomenon is ascribed to the universal photobleaching effect [28]. The dual effect of UV irradiation and air oxidation makes the bands decay quicker (Fig. 3D). Considering that argon protection is not convenient, results show that nitrogen and ascorbic acid solution could also protect the gel from being oxidized by oxygen (Fig. 3E & 3F). According to the above results, it is suggested that the gel imaging should be performed immediately after the staining to avoid the oxidation of the CuNCs. Otherwise, the gel is preferred to be stored in ascorbic acid solution or an oxygen-free environment if deferred or repeated observation is required. In

7 5 addition, like other dyes, the CuNCs-stained gel is better not exposed to light especially UV light for a long time. Despite the instability of CuNCs under air, protection is only required if the gel should be stored for repeated observation. In most cases that a gel is photographed for only once, the protection step can be neglected. In fact, except the gels shown in Figure 3, no protection is adopted for all the other experiments. The usability of CuNCs staining is studied by adopting different DNA fragments varying from core sequences of heterologous genes (HIV, HBV, and Hly), known template sequence (dsdna30/40), random sequence (dsdna21), sequences with specific structure (hairpin, and tweezer structure), DNA-RNA hybridization, and products of DNA amplification (HCR). The detailed sequences of these samples have been shown in the Supporting Information (Table S1 in the ESM). Results show that all the DNA samples can be well stained by the in situ synthesized fluorescent CuNCs (Fig. 4). However, it Figure 4 The fluorescence of different samples stained by CuNCs (left) and EtBr (right) in polyacrylamide gel (top) and solution (middle), respectively. From lane 1 to 9: dsdna21, dsdna30/40, HIV42, Hly44, HBV30, tweezer structure, products of HCR, hairpin structure, and DNA-RNA hybridization. The subscript number indicates the number of bases/base pairs. The concentration of all nucleic acids is fixed at 0.5 μm. Schematic illustration of the tweezer, HCR, and hairpin in lane 6-8 is also presented at the bottom. should be mentioned that in some cases the sensitivity of CuNCs staining is not as good as EtBr staining. Previous reports and our further study confirm that the sequences of DNA have effect on the fluorescence of CuNCs both in solution [22, 23] and in gel (Fig. S13 in the ESM). So, here as the molar quantities of the DNA samples are the same, the differences of the brightness of the CuNCs-stained bands should be ascribed to the sequence diversity. It is also noticed that the fluorescence in gel is not always in accord with that in solution. For example, a random sequence with 21 bp shows the brightest band in the gel (lane 1), while in solution, the corresponding fluorescence intensity is in the middle level. Further study shows that poly(t) can work as a template for the synthesis of fluorescent CuNCs in solution, while no corresponding bands can be observed in gel. In the case of poly(a), the phenomenon is just the reverse (Fig. S13). The reason for the disaccord is yet to be investigated. Finally, the skin toxicity test of CuNCs staining is performed in rats. The two reagents for the synthesis of CuNCs (copper sulfate and ascorbic acid) are known to be non-toxic in contact with skin. Copper sulfate is a microbicide, while ascorbic acid also known as vitamin c is even beneficial to human body. These two reagents have also been confirmed to be non-toxic in our skin toxicity test (Fig. S14 in the ESM). Furthermore, either the synthesizing CuNCs or the synthesized CuNCs have been spread onto the exposed skin of rats. In the case of the synthesizing CuNCs, copper sulfate and ascorbic acid are spread onto the skin once they are mixed together. Thus, this sample can mimic the staining solution that may be touched during the in situ synthesis of CuNCs in gel. As for the synthesized CuNCs, copper sulfate and ascorbic acid have been incubated together for 15 min before being spread to mimic the situation of taking out the gel. The local conditions of the treated skin area and the overall state of the rats are observed continuously in 30 days. The rats are finally dissected to study the changes of the liver. In all the Nano Research

8 6 Nano Res. cases of the observations, there is no abnormality (Fig. 5 and Fig. S14 in the ESM). So, it can be concluded Figure 5 The skin toxicity of synthesized CuNCs on adult male SD rats. The overall and local states of the rats after treated with CuNCs or H2O as control 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. preliminarily that the CuNCs staining is a green method. Finally, we would like to make a short discussion. The toxicity of conventional staining dyes should be mainly ascribed to their ability to bind to DNA and thus to affect the expression of genes in vivo. It seems impossible to solve this problem, since the interaction between a dye and a DNA strand is also necessary for staining in vitro. However, it should be noticed that another important reason for the toxicity of conventional dyes is that they may penetrate the skin and cell membrane to reach the cell nucleus. This reason offers a breakthrough for the development of a green staining method. Some ions and polar molecules (e.g. Cu 2+ and ascorbic acid) are known to pass through cell membrane through active transport. That is, cells reserve the right to decide whether they could enter or not. Also considering the skin barrier to these ions and polar molecules, they meet the requirement of the safety purpose. As a proof-of-concept, here we demonstrate that this way is accessible. In summary, we have developed a green staining method of DNA in polyacrylamide gel electrophoresis based on in situ synthesized fluorescent copper nanoclusters. After extensive trials, a simple procedure is finally established as follows. Firstly, the polyacrylamide gel is immersed in a solution containing CuSO4 for 15 min. Then, ascorbic acid is added to the solution and incubated for another 15 min to allow the in situ synthesis of DNA-templated fluorescent CuNCs. The above steps are repeated again; and the gel is ready for imaging under UV light. The comprehensive performances of the CuNCs-based staining method including the sensitivity, stability, usability, and safety have been studied. Favorable results are obtained, which allow the method to work as an alternative to the existing unsafe staining methods. It is particularly worth noting that the reagents adopted in this work and the whole staining process show possible high safety, which of course should be confirmed by more experimental evidences in the future. Though some limitation still exists (e.g. the sensitivity of CuNCs for some specific sequences is not as good as EtBr), it has the possibility to be conquered after further optimization and investigation in the future. The concept by in situ synthesizing of nanomaterials in biological system may also inspire application to other biotechnologies. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No , ), the National Science Fund for

9 7 Distinguished Young Scholars (Grant No ), the Natural Science Foundation of Shanghai (14ZR ), and the Innovation Program of Shanghai Municipal Education Commission (Grant No. 14YZ026). Electronic Supplementary Material: Supplementary material (experimental details, supplementary figures) is available in the online version of this article at References [1] Meyers, J. A.; Sanchez, D.; Elwell, L. P.; Falkow, S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. bacteriol. 1976,127, [2] Lee, J. D.; Huang, C. H.; Wang, N. W.; Lu, C. S. Automatic DNA sequencing for electrophoresis gels using image processing algorithms. J. Biomed. Sci. Eng. 2011, 4, [3] Ogier, J. C.; Son, O.; Gruss, A.; Tailliez, P. Delacroix-Buchet, A. Identification of the bacterial microflora in dairy products by temporal temperature gradient gel electrophoresis. Appl. Environ. 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10 Electronic Supplementary Material A green staining method of DNA in polyacrylamide gel electrophoresis based on in situ synthesized fluorescent copper nanoclusters Xiaoli Zhu 1, Hai Shi 1, Yalan Shen 1, Bin Zhang 1, Jing Zhao 1, and Genxi Li 1,2 ( ) Supporting information to DOI /s12274-****-****-* (automatically inserted by the publisher) 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 (MgCl2.6H2O), copper sulfate pentahydrate (CuSO4.5H2O), potassium chloride (KCl), sodium chloride (NaCl), sodium phosphate monobasic dihydrate (NaH2PO4.2H2O), disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O), 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 MgCl2, 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 Na2HPO4, 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 CuSO4 were added into a DNA solution successively and incubated together for 15 min in dark (dsdna30/40 Address correspondence to Genxi Li, genxili@nju.edu.cn. Nano Research

11 was adopted, sequences shown in the ESM, final concentrations of ascorbic acid, CuSO4, and DNA were 1 mm, 100 μm, and 0.5 μm, respectively). Several variables including ph, buffer, ion strength and temperature were 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 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 MgCl2, ph 7.5) containing 100 μm CuSO4 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 minutes 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 SD rats weighing ± 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 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 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'

12 3'-ATG AGT ATG CGA GTA CTG AAG-5' dsdna 20/30 dsdna 30/40 HIV 42 Hly 44 HBV 30 tweezer-struc tured DNA hairpin-struct ured DNA 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' 5'-TCT CCG CCT GCA AGT CCT AAG ACG CCA ATC GAA AAG AAA CAC GC-3' 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' HCR amplification DNA-RNA hybridization 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' Nano Research

13 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 dsdna30/40, 100 μm CuSO4, and 1 mm ascorbic acid). Photographs were taken 15 min after mixing all the reagents together.

14 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 Reference [1]: 10 mm MOPS buffer containing 150 mm NaCl. Other conditions are the same as that in Figure S Nano Research

15 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; MgCl2: 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 MgCl2, and 100 mm KCl, respectively. Other conditions are the same as that in Figure S1.

16 Figure S4 Effect of temperature on the formation of DNA-templated fluorescent CuNCs in 10 mm MOPS buffer solution containing 50 mm MgCl2 (ph 7.5). Other conditions are the same as that in Figure S Nano Research

17 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 MgCl2, 0.5 μm dsdna30/40, 100 μm CuSO4, and 1mM ascorbic acid) at 25 C for different reaction time.

18 Figure S6 Fluorescence spectra of the DNA-templated fluorescent CuNCs. Other conditions are the same as that in Figure S Nano Research

19 Figure S7 UV-vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl2, 100 μm CuSO4, and 1mM ascorbic acid) at 25 C in the absence of DNA template for different reaction time.

20 Figure S8 UV-vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl2, 100 μm CuSO4, and 1mM ascorbic acid) at 25 C in the presence of ssdna30 (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. Nano Research

21 Figure S9 UV-vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl2, 100 μm CuSO4, and 1mM ascorbic acid) at 25 C in the presence of dsdna30/40 (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.

22 Figure S10 UV-vis spectra of the CuNCs, which were synthesized in a MOPS buffer (10 mm MOPS, ph 7.5, 50 mm MgCl2, 100 μm CuSO4, and 1mM ascorbic acid) at 25 C in the presence of dsdna20/30 (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. Nano Research

23 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 MgCl2, at 25 C). Others are different from the optimized condition with single-variable, which has been marked over the ladder.

24 Figure S12 The ladder of DNA marker II with different molar quantities in polyacrylamide gel by using EtBr staining. Nano Research

25 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.

26 Figure S14 The skin toxicity of CuSO4, ascorbic acid, and synthesizing CuNCs on adult male SD rats. The overall and local states of the rats after treated with 100 μm CuSO4, or 1 mm ascorbic acid, or synthesizing CuNCs (CuSO4 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 [1] Rotaru, A.; Dutta, S.; Jentzch, E.; Gothelf, K.; Mokhir, A. Angew. Chem. Int. Ed., 2010, 49, Nano Research