Biostable L-DNA-Templated Aptamer-Silver Nanoclusters for Cell

Oct 31, 2016 - Yanjun Ding , Xingmei Li , Ceng Chen , Jiang Ling , Weichen Li , Yadong Guo , Jie Yan , Lagabaiyla Zha , Jifeng Cai ... Yue He , Bining...
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Biostable L‑DNA-Templated Aptamer-Silver Nanoclusters for CellType-Specific Imaging at Physiological Temperature Gui-Mei Han,†,‡,∥ Zhen-Zhen Jia,†,§,∥ Yan-Jun Zhu,†,‡,∥ Jia-Jia Jiao,†,§ De-Ming Kong,*,†,‡ and Xi-Zeng Feng*,†,§ †

State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, P R China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, Nankai University, Tianjin, 300071, P R China § The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science, Nankai University, Tianjin, 300071, P R China ‡

S Supporting Information *

ABSTRACT: The high susceptibility of the natural Dconformation of DNA (D-DNA) to nucleases greatly limits the application of DNA-templated silver nanoclusters (Ag NCs) in biological matrixes. Here we demonstrate that the L-conformation of DNA (L-DNA), the enantiomer of D-DNA, can also be used for the preparation of aptamer-Ag NCs. The extraordinary resistance of L-DNA to nuclease digestion confers much higher biostability to these NCs than those templated by D-DNA, thus making cell-type-specific imaging possible at physiological temperatures, using at least 100-times lower Ag NC concentration than reported D-DNA-templated ones. The L-DNA-templated metal NC probes with enhanced biostability might promote the applications of metal nanocluster probes in complex biological systems.

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DNA and other modified nucleic acids, such as locked nucleic acids and peptide nucleic acids, L-DNA is more stable in biological systems. This facilitates the interesting applications of L-DNA in different fields, including specific tags in PCR and microarrays,15,16 DNA nanocarriers,17 intracellular nanothermometers,18 biostable L-DNAzyme,14 Spiegelmers (from German Spiegel = mirror),19 Spiegelzymes,20 and asymmetric catalysis.21 It is reasonable to believe that L-DNA might be an appealing and promising option for the preparation of biostable DNA-templated aptamer-Ag NCs, thus enabling the application of Ag NCs in complex biological systems. Here we demonstrate for the first time that L-DNA can also be used for the synthesis of Ag NCs with bright fluorescence. Because the prepared Ag NCs are sufficiently stable against nuclease digestion, a very low concentration can meet the need for cell type-specific imaging at physiological temperature. One advantage of DNA-templated Ag NCs in bioimaging applications is that the aptamer sequence can be linked directly with the Ag NC nucleation sequence for one-stop synthesis of target-specific fluorescent aptamer-Ag NC hybrids. This overcomes the problem of difficult bioconjugation for Ag

ecause of the advantages of facile synthesis, ultrasmall size, tunable fluorescence emission, large Stokes shifts, high photostability, biocompatibility, and water solubility, DNAtemplated silver nanoclusters (Ag NCs) have attracted considerable attention in many fields.1−7 One such area is cell imaging. After examining the experimental conditions of the published studies, we have found that most experiments, especially those using aptamers as target recognition units, were conducted at low temperatures (e.g., 4 °C) or/and at high Ag NC concentrations (at least 10 μM).8−11 One reason might be that the used aptamer-Ag NCs were all prepared by using the natural D-conformation of DNA (D-DNA) as a template. It is well-known that D-DNA is susceptible to nucleases. The digestion of D-DNA by nucleases can result in quenched fluorescence and inhibition of the ability of aptamer-Ag NCs to recognize their targets. Nearly ubiquitous tissue distribution of nucleases in biological matrixes will greatly limit the applications of natural DNA-templated Ag NCs in biosensing and bioimaging. As the enantiomer of naturally occurring D-DNA (Scheme 1), the L-conformation of DNA (L-DNA) has identical physical properties in terms of solubility and duplex thermal stability12 but shows distinct characteristics including high resistance to nucleases and possible off-target effects caused by binding to single-stranded DNA-binding protein and/or partial hybridization with natural D-DNAs.13,14 Compared with natural D© XXXX American Chemical Society

Received: July 26, 2016 Accepted: October 31, 2016 Published: October 31, 2016 A

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Scheme 1. Schematic Representation of Application of L-DNA or D-DNA-Templated Aptamer-Ag NCs in Cell-Type-Specific Imaging

Figure 1. Resistance of DNA-templated aptamer-Ag NCs to DNase I. Fluorescence emission of (a) D-C10-AS1411 Ag NCs or (b) L-C10-AS1411 Ag NCs incubated with (1) 0 or (2) 20 U heat-inactivated DNase I or (3) 20 U active DNase I. The inserts show the results of the electrophoresis assay.

However, dramatically enhanced fluorescence emission was observed for D-C10-AS1411 Ag NCs compared to D-C10 Ag NCs. This is consistent with previous reports that dark DNAAg NCs can be turned on when placed in proximity to G-rich DNA sequences.9,24 Under the same conditions, and using the identical synthesis procedures, L-DNA can also be used as a template for the preparation of fluorescent Ag NCs. The L-C10AS1411 Ag NCs were also water-soluble and had similar size distribution and fluorescence emission properties, except that a slight red shift (∼4 nm) was observed for the peak emission wavelength compared to D-C 10-AS1411 Ag NCs. The fluorescence emission at the near-infrared range (λem = 660 nm) make the Ag NCs a suitable bioimaging probe for in vivo applications. The aim of our work was to obtain biostable Ag NCs by utilizing the resistance of L-DNA to nuclease digestion. To demonstrate this, the stabilities of the Ag NCs templated by DDNA or L-DNA were examined using deoxyribonuclease I (DNase I), an important enzyme that cleaves both single- and double-stranded DNAs and is responsible for at least 90% of the deoxynuclease activity in human plasma.25 After incubation

NCs with other target recognition molecules, which may not only disturb the stability and fluorescence properties of Ag NCs but also compromise the target recognition specificity and affinity of the recognition molecules.10 In this study, aptamer AS1411 was added to the 3′-end of a C-rich sequence (C10) to synthesize the fluorescent aptamer-Ag NC probe. The C-rich sequence was used as the DNA scaffold for the preparation of Ag NCs. AS1411 is a well-known aptamer for nucleolin, which is overexpressed on the surface of some malignant tumor cells.22,23 In this study, AS1411 plays two important roles. Besides its ability to recognize specific tumor cells, its G-rich sequence can also greatly enhance the fluorescence emission of the Ag NCs. First, a D-type DNA oligonucleotide (D-C10-AS1411, Table S1) was used for the synthesis of aptamer-Ag NC probe, and the morphology and fluorescence properties of the Ag NCs were compared with those of the D-C10 Ag NCs, using G-rich C10 oligonucleotide as a template (Figure S1). The transmission electron microscopy (TEM) image revealed that both D-C10 Ag NCs and D-C10-AS1411 Ag NCs are monodispersed and the majority of them are within the size range of 3−4 nm. B

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Figure 2. Fluorescence microscopic images of cancer cells (HeLa) and normal cells (NIH-3T3). The cells were treated with 100 nM L-C10-AS1411 Ag NCs or D-C10-AS1411 Ag NCs at 37 °C for 4 h, then the cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI).

with 20 U DNase I for 2 h, the fluorescence of D-C10-AS1411 Ag NCs was dramatically decreased (Figure 1a), which can be attributed to DNase I-triggered digestion of the Ag NC nucleation sequence (C 10 ) and the aptamer sequence (AS1411). Digestion of the C10 component can lead to the destruction of Ag nanoclusters, which can be demonstrated by DNase I-induced fluorescence quenching of D-C12 Ag NCs (Figure S2). Digestion of the AS1411 component reduces the fluorescence enhancement of Ag NCs by the G-rich sequence. More importantly, digestion of the aptamer sequence made the Ag NCs lose the ability to recognize nucleolin, thus preventing the ability of Ag NCs to image specific tumor cells. Under the same conditions, no obvious fluorescence signal change was observed in the presence of heat-inactivated DNase I, thus confirming that the fluorescence quenching of D-C10-AS1411 Ag NCs was caused by the digestion of D-DNA by active DNase I. Extraordinary resistance to DNase I digestion was observed for DNA-templated aptamer-Ag NCs when L-DNA was used instead of D-DNA. As shown in Figure 1b, DNase I has essentially no effect on the fluorescence emission of L-C10AS1411 Ag NCs, thus endowing the aptamer-Ag NCs with possibly higher potency for application in biological systems. The integrity of the L-DNA-templated aptamer-Ag NCs in the presence of DNase I was further verified by gel electrophoresis. In the absence of DNase I or in the presence of heat-inactivated DNase I, both D-DNA and L-DNAtemplated AgNCs gave a bright band in agarose gels. Incubation of D-C10-AS1411 Ag NCs with active DNase I resulted in the disappearance of the bright band, indicating that the integrity of the D-DNA-templated aptamer-Ag NCs had

been destroyed. By contrast, a distinct band with similar mobility and brightness was still observed for L-C10-AS1411 Ag NCs, thus demonstrating that L-DNA-templated aptamer-Ag NCs remained intact even in the presence of active DNase I. Similar results were obtained when DNase I was replaced by exonuclease I (Exo I), a 3′ → 5′ exonuclease that can be used specifically for single-stranded DNA degradation (Figure S3). After demonstrating the high nuclease resistance of L-DNAtemplated aptamer-AgNCs, we investigated the feasibility of using L-C10-AS1411 Ag NCs for cell-type-specific imaging at physiological temperatures. After incubation of L-C10-AS1411 Ag NCs either with cancer cells (HeLa) or with normal cells (NIH-3T3) in the absence of any transfection reagent at 37 °C, fluorescence microscopic images were obtained. As illustrated in Figure 2, the Ag NC signal was displayed in red. HeLa cells incubated with L-C10-AS1411 Ag NCs displayed bright fluorescence. However, almost no fluorescence signal was observed for NIH-3T3 cells under the same conditions. Benefiting from the greatly enhanced biostability of L-C10AS1411 Ag NCs, a concentration of 100 nM L-DNA-templated aptamer-Ag NCs can work well for HeLa cell-specific imaging at 37 °C. This concentration is about 100 to 2000 times lower than that used in studies on cell imaging using DNA-templated aptamer-Ag NCs.9,11 Low aptamer-Ag NC concentration ensures the maintenance of good cell morphology, which is superior to those in works using high Ag NC concentrations. There are two possible mechanisms for the internalization of LC10-AS1411 Ag NCs in HeLa cells. One is via nucleolinmediated endocytosis (Scheme 1),8 which is supported by the fact that nucleolin is expressed at high levels on HeLa cells but not on NIH-3T3 cells. It should be noted that the well-known C

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DNA aptamer to natural D-DNA one, makes L-DNA templated aptamer-Ag NCs more suitable for in vivo targeted applications. They may thus help us understand the roles of specific proteins in cell processes and human diseases in more detail. Benefiting from greatly enhanced biostability, tumor cell-type-specific imaging is achieved at physiological temperature using very low concentrations of L-DNA-templated aptamer-Ag NCs. Compared to organic fluorophores and other luminescent nanoprobes (e.g., quantum dots), Ag NCs show many superior features such as tunable luminescent properties, small sizes, high photostability, nontoxicity, and so on.27 L-DNA-dependent enhancement of biostability will certainly confer this kind of luminescent probe with much wider application perspectives. With the discovery of the Spiegelmer technology, more and more L-DNA-based aptamers targeting at natural biomolecules have been reported,19,28 thus implying that the proposed LDNA-based aptamer-Ag NCs strategy might have broad prospect in bioapplications. Besides Ag NCs, DNA has been successfully used for the preparation of other metal nanoclusters (e.g., Au NCs and Cu NCs),29−31 the biostability of these metal nanoclusters might be also improved by using LDNA as templates. Overall, this work offers an exciting method to design biostability-enhanced, DNA-templated metal nanocluster probes, which will not only provide new schemes to utilize L-DNA for bioanalysis and bioimaging but also broaden the applications of DNA-templated metal nanoclusters in complex biological matrixes.

AS1411 aptamer targeting at nucleolin is built by natural DDNA. The significant different L-C10-AS1411 Ag NCs internalization in HeLa and NIH-3T3 cells suggests that LDNA-based AS1411 might also work well for nucleolin. The other possible mechanism is via AS1411-induced micropinocytosis that was proposed by Reyes-Reyes et al.22,26 In this mechanism, nucleolin also plays important roles since it is necessary for induced micropinocytosis and AS1411 uptake. Under the same conditions, neither HeLa nor NIH-3T3 cells could give observable fluorescence signal upon treatment with D-C10-AS1411 Ag NCs, which might be attributed to poor biostability of D-C10-AS1411 AgNCs. Biodegradation of the DDNA-templated aptamer-Ag NCs by nucleases not only leads to quenched fluorescence emission but also reduces the aggregation of them on the surface of HeLa cells. Certainly, nucleolin-mediated Ag NCs uptake is also impaired. Even if a certain amount of D-C10-AS1411 Ag NCs are successfully internalized into HeLa cells, the presence of nucleases in cell interior might also lead to the degradation of the D-DNAtemplated aptamer-Ag NCs. To verify above experimental results, inductively coupled plasma mass spectrometry (ICPMS) and flow cytometry experiments were conducted. ICPMS analysis showed that about 2.74 and 3.45 ng silvers were absorbed by 10 000 HeLa cells when D-C10-AS1411 and L-C10-AS1411 Ag NCs were used, respectively. Different uptake efficiencies might be due to partial degradation of D-C10-AS1411 Ag NCs before internalization in cells. Although D-C10-AS1411 Ag NCs have lower cell internalization efficiency than L-C10-AS1411 Ag NCs, a certain amount of D-C10-AS1411 Ag NCs was still found to be internalized in HeLa cells. Thus, the much poorer cell imaging capability of D-C10-AS1411 Ag NCs than L-C10-AS1411 Ag NCs might also be attributed to their further degradation in cells. The experimental results of flow cytometry assays also demonstrated that L-C10-AS1411 Ag NCs have better internalization efficiency in HeLa cells than D-C10-AS1411 Ag NCs, and aptamer-Ag NCs uptake capability of HeLa cells was much better than NIH-3T3 cells (Figure S4). The cytotoxicity of both L-C10-AS1411 Ag NCs and D-C10AS1411 Ag NCs was investigated by using the standard MTT assay (Figure S5). Since Ag+ is highly toxic to cells, free Ag+ must be removed before the assay. After incubation of HeLa cells with L-C10-AS1411 Ag NCs or D-C10-AS1411 Ag NCs for 24 h, more than 90% of cells were alive even when the Ag NC concentration was as high as 500 nM, which is much higher than that used in cell imaging studies. After extending the incubation time to 72 h, nearly 90% of the cells were still viable. These results show that the use of non-natural L-DNA does not increase the cytotoxicity of DNA-templated aptamer-Ag NCs. The aptamer-Ag NCs have very low toxicity for living cells and, thus, might be a good candidate as a specific probe for cell imaging. In previous work,11 DNA-templated Ag NCs were reported to show cytotoxicity to living cells. One reason might be the high concentration of used Ag NCs (0.19 mM). In this work, low concentration can ensure negligible cytotoxicity of LDNA-templated aptamer-Ag NCs to living cells and biological tissues. In summary, we have described the synthesis of the first example of L-DNA-templated aptamer-AgNCs. Compared with the natural D-DNA-templated Ag NCs, L-DNA-templated aptamer-Ag NCs have much higher nuclease resistance. This advantage, combined with fluorescence emission in the nearinfrared range and the similar target recognition specificity of L-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b02871. Full experimental details, characterization and biostability measurements of DNA-templated aptamer-Ag NCs (Figures S1−S3), flow cytometry analysis of aptamerAg NCs uptake by cells (Figure S4), and cytotoxicity assay of the prepared aptamer-Ag NCs (Figure S5) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ∥

G.-M.H., Z.-Z.J., and Y.-J.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research P r o g r a m o f C h i n a ( G r a n t s 2 0 11 C B 7 0 7 70 3 a n d 2015CB856500), the National Natural Science Foundation of China (Grant No. 21322507), and the National Natural Science Foundation of Tianjin (Grant No. 16JCYBJC19900).

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