Elsevier

Talanta

Volume 169, 1 July 2017, Pages 57-63
Talanta

Label-free and sensitive assay for deoxyribonuclease I activity based on enzymatically-polymerized superlong poly(thymine)-hosted fluorescent copper nanoparticles

https://doi.org/10.1016/j.talanta.2017.03.047Get rights and content

Highlights

  • A green, facile, low-cost, label-free and ultrasensitive assay for DNase I activity.

  • A seamless integration of DNase I digestion and TdT polymerization.

  • TdT-polymerized superlong poly T ssDNA (>500 mer) for fluorescent CuNPs formation.

  • A limit of detection as low as 0.02 U/mL was realized for DNase I analysis.

  • DNase I in diluted serum was successfully detected with superlong poly T-CuNPs.

Abstract

Deoxyribonuclease I (DNase I) is an important physiological indicator and diagnostic biomarker, but traditional methods for assessing its activity are time-consuming, laborious, and usually radioactive. Herein, by effectively combining the special functions of DNase I and terminal deoxynucleotidyl transferase (TdT), a simple, green, cost-effective, label-free and ultrasensitive assay for DNase I activity has been constructed based on superlong poly(thymine)-hosted copper nanoparticles (poly T-CuNPs). In this strategy, a 3′-phosphorylated DNA primer is designed to block TdT polymerization. After addition of DNase I, the primer could be digested to release 3′-hydroxylated fragments, which could further be tailed by TdT in dTTP pool with superlong poly T ssDNA for CuNPs formation. Fluorescence measurements and gel electrophoresis demonstrated its feasibility for DNase I analysis. The results indicated that with a size of 3–4 nm, the CuNPs templated by TdT-polymerized superlong poly T (>500 mer) had several advantages such as short synthetic time (<5 min), large Stokes shift (~275 nm) and intense red fluorescence emission. Under the optimal conditions, quantitative detection of DNase I was realized, showing a good linear correlation between 0.02 and 2.0 U/mL (R2=0.9928) and a detection limit of 0.02 U/mL. By selecting six other nucleases or proteins as controls, an excellent specificity was also verified. Then, the strategy was successfully applied to detect DNase I in diluted serum with a standard addition method, thus implying its reliability and practicability for biological samples. The proposed strategy might be promising as a sensing platform for related molecular biology and disease studies.

Graphical abstract

By seamlessly integrating the special functions of deoxyribonuclease I (DNase I) and terminal deoxynucleotidyl transferase (TdT), a novel fluorescent sensor for DNase I activity has been developed based on superlong poly(thymine)-hosted copper nanoparticles (poly T-CuNPs). The strategy is green, facile, cost-effective, label-free and highly-sensitive, thus achieving a limit of detection as low as 0.02 U/mL and potential applicability to biological samples.

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Introduction

As the first enzyme specific to DNA, deoxyribonuclease I (DNase I, EC 3.1.21.1) can endonucleolytically hydrolyze phosphodiester linkages of DNA to release di- and oligonucleotides with 5′-phosphate (5′-PO3) and 3′-hydroxyl (3′-OH) groups [1]. It is ubiquitously expressed in mammalian tissues and fluids, especially abundant in pituitary gland and pancreas [2]. Without nucleotide sequence specificity, DNase I can act on various substrates including single-stranded DNA, double-stranded DNA, chromatin and RNA/DNA hybrids, and thus plays essential roles in DNA repair, waste management and cell apoptosis [3], [4]. Moreover, the variation of DNase I activity has also been found to be closely related to diseases such as systemic lupus erythematosus (SLE), cancer, and cardiovascular diseases [5], [6], [7]. For example, DNase I activity was demonstrated to be lower in blood of cancer patients, which might be the reason for increased quantity and integrity of cell-free circulating DNA [8], [9]. Hence, the detection of DNase I activity, an important physiological indicator and a useful diagnostic biomarker, is critically essential.

Traditional methods for assessing nuclease activity, such as high performance liquid chromatography (HPLC) [10], enzyme-linked immunosorbent assay (ELISA) [11], etc., are time-consuming, laborious, discontinuous and usually radioactive. To overcome the defects, several methods have recently been developed for DNase I detection by using different signal output techniques, including colorimetric analysis [12], [13], electrochemical assays [14] and fluorescent methods [15]. Thereinto, fluorescent signal-based assays have attracted much attention due to their advantages of high sensitivity, fast response, easy operation, no radiation exposure and versatility [16], [17]. For example, single radial enzyme diffusion (SRED), a simple and sensitive assay for DNase I, has been developed by using ethidium bromide (EB) or SYBR Green I to stain digested DNA products [18]. However, SRED is time-consuming. Worse still, EB is toxic to biological processes (DNA replication and transcription), and is considered as a strong mutagen with high carcinogenicity [19]. Another method is a fluorescence resonance energy transfer (FRET)-based hairpin-shaped DNA probe with partially phosphorothioate-modified backbone, which realized real-time imaging of DNase I activity in living cells [20]. Also, magnetic particles [21] and graphene oxide (GO) [15], [22] have respectively been combined with DNA probes for specific and sensitive analysis of DNase I. Nevertheless, in the above methods, special sequence design or labeling of DNA probes is commonly required. Also, complex preparation and modification of nanomaterials is usually involved. Therefore, it is still necessary to develop a simple, green, cost-effective, label-free and sensitive assay for DNase I activity.

Recently, a novel fluorescent nanomaterial, single-stranded poly(thymine) (poly T) DNA-templated copper nanoparticles (poly T-CuNPs), has been found and studied in our group [23]. Based on high affinity between Cu2+ and thymine [24], [25], poly T-CuNPs can form through chemical reduce of thymine-complexed Cu2+ to Cu° by reducing agents along the poly T DNA scaffold. The synthesis process is simple, convenient, highly-efficient, and extremely fast (within 5 min at room temperature). The produced poly T-CuNPs can emit strong red fluorescence (λem=615 nm) under excitation of a maximum wavelength of 340 nm, due to the quantum size effect. Such a MegaStokes shifting (275 nm) is particularly advantageous for poly T-CuNPs to detect targets in complex biological media. Moreover, because Cu is an essential micronutrient for humans and animals, poly T-CuNPs should be biologically safer than traditional toxic organic dyes and other heavy-metal nanomaterials [26], [27], [28]. As a label-free, low-cost and green analysis tool, poly T-CuNPs have thus been applied to measure various targets, including ions [29], [30], [31], molecules [32], [33], [34], [35], [36], [37] and proteases [38], [39]. However, because the fluorescence of poly T-CuNPs is highly dependent on the length of templates, sensitivity may be greatly limited by directly using commercially-purchased poly T templates (usually dozes of bases). Recently, terminal deoxynucleotidyl transferase (TdT), a template-independent polymerase that catalyzes the addition of deoxynucleotides to the 3′-OH of DNA [40], [41], has been utilized to generate superlong poly T sequences (hundreds of bases) as templates for CuNPs formation [42], [43], [44]. The strategy is expected to significantly improve the fluorescence of poly T-CuNPs, and has been used to detect several targets, such as DNA [45], TdT [46], polynucleotide kinase (PNK) [47], etc. But as far as we know, its application in DNase I assay has not been reported yet.

In this paper, by smartly combining the special functions of DNase I and TdT, a simple, green, label-free and highly-sensitive method for assessing DNase I activity has been established. As shown in Scheme 1, a primer DNA is phosphorylated at 3′ terminus (DNA-3′-PO3) to block the polymerization of TdT, by utilizing the property that only DNA with 3′-OH can serve as the substrate of TdT. However, when target DNase I exists, the primer DNA-3′-PO3, serving as its substrate, can be randomly cleaved, thus producing several short oligonuleotides with 3′-OH. Then, the digested DNA products with 3′-OH can be tailed with a superlong poly T sequence by TdT in the presence of 2′-deoxythymidine 5′-triphosphate (dTTP). And finally, the superlong poly T tails can be used to template the formation of fluorescent CuNPs for DNase I activity analysis. The strategy is facile, in which the primer DNA probe has no requirements for base sequence and dye labeling. Furthermore, a preliminary amplification might be realized with the function of DNase I that one primer DNA molecule can generate several DNA molecules with 3′-OH to initiate the following polymerization. And a further combination with the TdT-mediated superlong poly T-CuNPs amplification process might promise a label-free and highly-sensitive assay.

Section snippets

Chemicals and materials

Deoxyribonuclease I (DNase I) and terminal deoxynucleotidyl transferase (TdT) were purchased from New England Biolabs, Ltd. (Beijing, China). The primer DNA with 3′-PO3 (DNA-3′-PO3, 5′-AAC TAT GCA ACC TAC TAC CTC T-PO3-3′), control DNA with 3′-OH (DNA-3′-OH, 5′-AAC TAT GCA ACC TAC TAC CTC T-OH-3′) and 2′-deoxythymidine 5′-triphosphate (dTTP) solutions were obtained from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Sodium ascorbate was purchased from Aladdin Industrial Inc.

Feasibility of the superlong poly T-CuNPs based strategy for DNase I analysis

The feasibility of the poly T-CuNPs based DNase I assay was firstly investigated through fluorescence measurements. As shown in Fig. 1, six groups of DNA samples were individually used to synthesize CuNPs by adding Cu2+ and ascorbate. The primer DNA was blocked by 3′-PO3 (DNA-3′-PO3) and the control DNA was designed with 3′-OH (DNA-3′-OH). In the absence of DNase I, DNA-3′-PO3 and DNA-3′-OH were respectively used as the substrate of TdT to generate poly T ssDNA for CuNPs synthesis. The signal

Conclusion

A simple, green, cost-effective, label-free and ultrasensitive method for DNase I assay has been developed based on the TdT-mediated synthesis of superlong poly T-CuNPs. The strategy makes full use of the substrate specificity of TdT that only DNA with 3′-OH could initiate the template-independent polymerization, and smartly combines the function of DNase I in generating DNA products with 3′-OH. Just in the presence of DNase I, TdT could polymerize dTTP into superlong poly T ssDNA (>500 mer) to

Acknowledgment

This work was supported by the National Natural Science Foundation of China (Grants 21190044, 21305038, 21575037, 21521063 and 21675046), and the Hunan Provincial Natural Science Foundation (Grant 2015JJ3044).

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