Terminal protection of small molecule-linked ssDNA for label-free and highly sensitive colorimetric detection of folate receptor biomarkers

Abstract

Small molecule/protein interactions have a key role in drug discovery, clinic diagnosis and protein–metabolite interactions in biology. By using the specific interaction between folic acid (FA) and folate receptor (FR) as a model, the development of a label-free and sensitive colorimetric approach for the detection of the FR biomarker is described. The sensing approach relies on the coupling of the FR-induced terminal protection of FA-linked ssDNA strategy with significant signal amplification by self-assembled DNAzyme polymers. The FR binds to the FA-ssDNA and protects the FR/FA-ssDNA from digesting by exonuclease I. The terminal protected ssDNA further triggers autonomous self-assembly of two G-quadruplex sequence-containing hairpin DNAs into DNAzyme polymers, which result in intensified color change of the probe solution for label-free and highly sensitive colorimetric detection of FR. The terminal protection mechanism and the self-assembly formation of the DNAzyme polymers are characterized by using polyacrylamide gel electrophoresis, and the sensing parameters are optimized as well. Under optimal experimental conditions, the detection limit of 0.35 pM for FR can be obtained by using a UV-Vis spectrophotometer and the presence of as low as 5 pM of FR can be directly visualized by the naked eye. The developed method is also selective and can be applied to detect FR in serum samples, which makes this approach a sensitive platform for sensing different types of small molecule/protein interactions.

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Introduction

Specific interactions between small organic molecules and the corresponding affinity proteins regulate most physiological processes of organisms,^1,2 and the detection of these types of interactions plays important roles in clinical diagnosis, chemical genetics and drug screening.^3,4 Current methods available for the analysis of small molecule/protein interactions include affinity chromatography,^5,6 kinetic capillary electrophoresis,^7,8 surface plasmon resonance (SPR),⁹ fluorescence resonant energy transfer^10,11 and protein-fragment complementation assays.^12,13 Although these approaches have been demonstrated to be effective assay tools for the investigation of small molecule/protein interactions, the involvement of sophisticated instruments and cumbersome assay procedures with compromised sensitivity has limited their wide applications. Therefore, it is highly demanded for researchers to develop cost-effective and sensitive assay strategies for the monitoring of small molecule/protein interactions.

The terminal protection of small molecule-linked DNA reported by the Jiang group¹⁴ has emerged as an ideal approach for detecting interactions between small molecules and their protein receptors. The assay mechanism was based on the employment of small molecule-tethered DNA as probes to bind to the target proteins through small molecule/protein interactions, which prevented the ssDNA from digesting by exonuclease I (Exo I) and generated electrochemical current responses for detecting the target proteins. The ssDNA involved in the assay protocol can not only offer the capability of selective capture of the target proteins but also provide versatile subsequent signal amplification means for achieving high sensitivity due to the nucleic acid nature of the ssDNA. By following this terminal protection mechanism, a number of methods for protein detections have been recently reported in connection with various electrochemical^15,16 and fluorescent^17–19 signal transduction techniques. Despite these advances, the development of simple, sensitive, homogeneous and label-free approaches for the detection of small molecule/protein interactions has been rarely reported. The electrochemical methods for the detection of small molecule/protein interactions commonly require the immobilization of the ssDNA on the electrode surface, while the fluorescent approaches involve the conjugation of the fluorescent tags, which potentially increase the complexity and cost for the assay methods. Therefore, the exploration of a label-free and sensitive approach without using any complex instrument will facilitate the monitoring of small molecule/protein interactions.

Colorimetric detections have gained increasing attention due to the extreme simplicity, low cost and especially the macroscopically observable characteristics when encountering the target analyte.^20,21 Gold Nanoparticles (AuNPs), with unique optical properties associated with their surface plasmon resonance and high extinction coefficients, are perfectly suitable for colorimetric assays.^22,23 However, the AuNP-based colorimetric assays are limited by their compromised sensitivities,^24,25 time consuming (about 20–48 h for nanoparticles preparation and probe-conjugation)²⁶ procedures and susceptibility to sensing environments with false positive signals (ionic strength, temperature, etc.).^27,28 With regards to these challenges in AuNP-based colorimetric detection approaches, a new class of functional nucleic acid probes, the peroxidase mimicking DNAzymes, have been increasingly used recently. The DNAzyme contains a complex of hemin and a single-stranded guanine-rich nucleic acid (G-quadruplex), which can catalyze H₂O₂-mediated oxidization of 2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) to generate a blue-green colored product, ABTS˙⁻ (λ_max = 421 nm).^29,30 This class of DNAzymes offers significant advantages, including easy synthesis, thermal stability, and has been extensively used as biocatalytic labels in amplified biosensing of proteins,³¹ metal ions³² and small molecules.³³

Herein, based on terminal protection of small molecule-linked ssDNA and significant signal amplification by self-assembled hemin/G-quadruplex DNAzyme polymers, we report on a label-free, homogenous and highly sensitive colorimetric platform for the detection of folate receptor (FR), a biomarker associated with numerous malignancies, including myelogenous leukemias, ovarian, lung, kidney and breast cancers.^34–36 The association of FR with the folic acid (FA)-linked ssDNA prevents the ssDNA from digesting by Exo I, and the protected ssDNA triggers the hybridization chain reaction (HCR) self-assembly formation of numerous DNAzymes, which catalyze the conversion of colorless ABTS to green colored ABTS˙⁻ and lead to significant color change of the probe solution for colorimetric detection of FR down to the sub-picomolar level.

Experimental

Apparatus

A Shimadzu 2450 UV-Vis spectrophotometer (Shimadzu, Japan) was used to acquire the UV-Vis absorption spectra at room temperature in all experiments and a canon EOS 550D camera was used to take all photographs.

Reagents and materials

Folic acid (FA), N-hydroxysulfosuccinimide (Sulfo-NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), bovine serum albumin (BSA), streptavidin (SA), mouse immunoglobulin G (IgG) were purchased from Sigma (St. Louis, MO). Folate receptor (FR) was obtained from Beijing Biosynthesis Biotechnology CO., Ltd (Beijing, China). Exo I and all oligonucleotides with the following sequences were purchased from Sangon Biotechnology Co. Ltd (Shanghai, China). H1: 5′-TGGGTTCTTCTTCCACAAATTCATGTTAAGAGGTTGAATTTGTGGGTGGGCGGGA-3′; H2: 5′-GGGCGGGATGGGTTGAATTTGTGGAAGAAGATCAAATTCAACCTCTTAACTGGGT-3′; ssDNA probe: 5′-ATGAATTTGTGGAAGAAGA-NH₂-3′. Hemin, [tris(hydroxymethy-l)aminomethane] (Tris), 2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS), H₂O₂, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt (HEPES) were purchased from Aladdin Reagents (Shanghai, China). The stock solution of hemin (5 μM) was prepared in dimethylsulfoxide (DMSO) and stocked in the dark at −20 °C. All reagents were of analytical grade and used without further purification.

Preparation of the FA-ssDNA probes

FA was conjugated to the 3′-NH₂ of the ssDNA probes by using the succinimide coupling (EDC-NHS) approach.³⁷ Briefly, ssDNA probes (20 μM) were incubated with 2 mM folate, 1 mM EDC and 5 mM Sulfo-NHS in 10 mM PBS (pH 7.4) for 2 h at 37 °C in the dark. This was followed by further purification of the mixture to obtain the FA-ssDNA by using the Illustra MicroSpin G-25 Columns (GE Healthcare), which were designed for rapid purification of DNA by the process of gel filtration and were ideal for the purification of oligonucleotides or small DNA fragments following synthesis or a labeling reaction.³⁸

Label-free and amplified colorimetric detection of FR

The FA-ssDNA probes (10 μL, 0.5 μM) were incubated with 10 μL of different concentrations of FR in Tris-buffer (25 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl₂, pH 7.4) at 37 °C for 60 min in dark to allow complete interaction between FR and FA-ssDNA. Exo I (10 U) was then added to the mixture at 37 °C for 30 min to digest the un-protected FA-ssDNA, followed by the termination of the digestion reaction with the addition of 20 mM EDTA. After that, the resulting solution was mixed with H1 (10 μL, 2 μM) and H2 (10 μL, 2 μM) in Tris-buffer with total volume of 60 μL for 6 h at room temperature to proceed the self-assembly process.³⁹ Then, 10 μL (5.0 μM) hemin in 2× HEPES buffer (50 μM HEPES, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, 2% DMSO, pH 7.4) was added to the solution, and the mixture was incubated at room temperature for 60 min to form the hemin/G-quadruplex DNAzymes. Finally, ABTS²⁻ and H₂O₂ were added to the mixture to reach final concentrations of 6 mM and 2 mM, respectively, at room temperature, and photographs of the solutions were taken after 5 min of color development.

Native polyacrylamide gel electrophoresis (PAGE)

The sample solutions were subjected to electrophoresis analysis on the 16% non-denaturing polyacrylamide gel. Electrophoresis was carried out in 1× TBE (pH 8.3) at a 100 V constant voltage for 50 min. The gels were stained with SYBR Gold (Invitrogen, Inc.) for 20 min and photographed under a UV illumination with a digital camera.

Results and discussion

Our label-free, homogenous and sensitive colorimetric approach for FR detection based on the terminal protection strategy and self-assembled hemin/G-quadruplex DNAzymes is illustrated in Scheme 1. This protocol involves the FA-linked ssDNA probes, Exo I and two metastable hairpin DNAs (H1 and H2). The G-quadruplex-forming sequence is split into two segments (the blue a and d regions in the hairpin structures), which are respectively incorporated into the 3′ and 5′ terminus and partly hybridized with the stem of the hairpin DNA to prevent the formation of G-quadruplex in the folded hairpin DNA structures. In the absence of the FR target, the FA-ssDNA can be efficiently digested into mono-nucleotides by Exo I, which catalyzes the removal of nucleotides from ssDNA in the 3′ to 5′ direction. The hairpin DNAs remain folded in the solution to avoid the formation of DNAzymes upon subsequent addition of hemin because of the G-quadruplex-forming sequences are locked in the stems. On the contrary, when FR is incubated with the FA-ssDNA probes, it binds to the FA moieties of the FA-ssDNA at the 3′ termini with high affinity and protects the FA-ssDNA from digesting by Exo I due to the steric hindrance of the bound FR proteins, which prevent Exo I from approaching and cleaving the phosphodiester bonds adjacent to the 3′ termini of the ssDNA. Since the binding has no effect on the nucleic acid property of the ssDNA, the hybridization between the protected ssDNA and H1 will therefore not be affected. As a result, the terminal protected ssDNAs hybridize with and unfold H1 to unlock the G-quadruplex forming sequences in H1. Similarly the single-stranded region of the unfolded H1 can hybridize with and unfold H2 to unlock the G-quadruplex forming sequences in H2. Again, the unfolded H2 hybridizes with H1 to assemble H1 and H2 into DNA polymers in a HCR fashion, which brings the segments of the G-quadruplex forming sequences into proximity. Subsequent incubation of the DNA polymers with hemin leads to the formation of numerous hemin/G-quadruplex DNAzymes, which cause significantly intensified green color transition of the probe solution (in the presence of H₂O₂ and ABTS) for achieving highly sensitive colorimetric detection of FR.

Scheme 1 Illustration of the label-free and sensitive colorimetric detection of FR based on the terminal protection strategy and self-assembly of hemin/G-quadruplex DNAzyme polymers.

The FR-induced terminal protection and HCR formation of the DNA polymers were first verified by native PAGE (16% gel). As shown in Fig. 1A, the FA-ssDNA (2 μM) alone exhibits a clear band (lane a), and the incubation of FA-ssDNA with FR (1 μM) leads to the appearance of a new band with lower electrophoretic mobility (lane b), indicating the successfully association of FR with the FA moieties of the FA-ssDNA. Subsequent addition of Exo I (20 U) to the mixture of FR and FA-ssDNA results in the disappearance of the band corresponding to the FA-ssDNA (lane c vs. b), which suggests the digestion of the un-protected FA-ssDNA by Exo I. After the deactivation of Exo I and further incubation of the mixture with H1 (5 μM) and H2 (5 μM), bands with various electrophoretic mobility, which correspond to the characteristic bands of HCR,⁴⁰ are observed, suggesting the self-assembly formation of the DNA polymers triggered by the FR-protected ssDNA. The proof-of-concept application of this strategy for amplified colorimetric detection of FR was also evaluated. According to Fig. 1B, the mixture of H1 and H2 exhibits minimal UV-Vis absorption (curve a) with the addition of hemin and subsequent incubation with ABTS and H₂O₂ because the G-quadruplex-forming sequences are initially locked in the stems of the hairpin DNAs. The incubation of FA-ssDNA (0.5 μM) with Exo I (20 U) in the absence of FR, followed by further incubation with H1 (2 μM) and H2 (2 μM), shows slight increase in UV-Vis absorption (curve b vs. a) compared with that of the mixture of H1 and H2, indicating that the FA-ssDNA is digested by Exo I and is unable to trigger HCR between H1 and H2. However, when FR (30 pM) is first incubated with FA-ssDNA, followed by further incubation with Exo I, deactivation of Exo I and the addition of H1 and H2, significant increase in UV-Vis absorption is observed (curve c vs. b). Such increase is basically due to the formation of numerous DNAzymes by self-assembly of H1 and H2 with the FR-protected ssDNA as discussed previously. The results here clearly demonstrate that the binding of FR to the FA-ssDNA can protect the FA-ssDNA from digesting by Exo I and the protected ssDNA can trigger HCR amplification to substantially intensify the color change of the probe solution for sensitive detection of FR.

Fig. 1 (A) Native PAGE analysis of different reaction mixtures: (a) FA-ssDNA only (2 μM); (b) FA-ssDNA (2 μM) and FR (1 μM); (c) FA-ssDNA (2 μM), FR (1 μM) and Exo I (20 U); (d) FA-ssDNA (2 μM), FR (1 μM), Exo I (20 U), H1 (5 μM) and H2 (5 μM). (B) Typical UV-Vis absorption spectra of (a) H1 (2 μM) and H2 (2 μM); (b) FA-ssDNA (0.5 μM), Exo I (20 U), H1 (2 μM) and H2 (2 μM); (c) FA-ssDNA (0.5 μM), FR (30 pM), Exo I (20 U), H1 (2 μM) and H2 (2 μM). FR was first incubated with FA-ssDNA for 60 min, followed by further incubation with Exo I (40 min) and deactivation of Exo I. H1 and H2 were then added to the mixture and incubated for 6 h, followed by the incubation with hemin (5.0 μM) for 60 min. Finally, ABTS and H₂O₂ were added and incubated for 5 min.

In order to achieve optimal analytical performance, the effects of the amount of Exo I and Exo I-catalyzed digestion time on the UV-Vis absorption of the probe solutions were investigated. The amount of Exo I was optimized within the range of 2–14 U. As shown in Fig. 2A, by incubating the FA-ssDNA probes (0.5 μM) with different amounts of Exo I at 37 °C for 40 min, the UV-Vis absorption intensity of the solution gradually decreases with increasing amount of Exo I until 10 U and then reaches a plateau. Therefore, 10 U was selected as the optimized amount of Exo I. The effect of Exo I digestion time was investigated by monitoring the UV-Vis absorption of the solution in the absence of FR. From Fig. 2B, we can see that the absorption intensity decreases rapidly with the augment of the digestion time and there is no significant decrease after 30 min, indicating complete digestion of FA-ssDNA by Exo I. The Exo I digestion time was thus fixed at 30 min for subsequent experiments.

Fig. 2 Effects of (A) the amount of Exo I and (B) Exo I cleavage time on the UV-Vis absorption intensity of the FA-ssDNA probe solution. Error bars, SD, n = 3.

Under optimal conditions, the application of the proposed terminal protection strategy for quantitative detection of FR was evaluated. From Fig. 3A, we can clearly see that the presence of FR from 0 to 100 pM results in gradual increase of the UV-Vis absorption and the intensity at 421 nm exhibits a linear correlation to the corresponding concentration of FR with a coefficient of correlation of 0.998. According to the calibration plot in the inset of Fig. 3A, the detection limit for FR is calculated to be 0.35 pM based on the standard 3σ rule. As shown in Fig. 3B, it is clear that the color change of the probe solution is gradually intensified with increasing concentration of FR from 0 to 100 pM by naked eye, which is consistent with the UV-Vis absorption in Fig. 3A. Compared with the blank test (Fig. 3B, b vs. a), as low as 5 pM of FR can be directly visualized by naked eye. Such visual detection limit for FR is comparable to those methods based on electrochemical⁴¹ or fluorescent transduction strategies,⁴² which further demonstrates the significant signal amplification capability of the developed method. The dramatic signal amplification is closely related to the design of the hairpin DNAs, in which each hairpin DNA (H1 or H2) contains one locked G-quadruplex-forming sequence, and after HCR, the amount of the DNAzymes is significantly enhanced compared to other common approach for HCR formation of the DNAzymes.⁴³

Fig. 3 (A) Typical UV-Vis absorption spectra of the colorimetric method for the detection of different concentrations of FR. From bottom to top: 0, 1, 5, 10, 25, 50, 75, 100 pM. Inset: the corresponding calibration plot of the concentration of the FR vs. the absorbance intensity. Error bars: SD, n = 3. The amount of Exo I (10 U); 30 min incubation time for Exo I. Other conditions, as in Fig. 1B. (B) Photograph for visual detection of FR at different concentrations: (a) 0, (b) 5, (c) 10, (d) 25, (e) 50, (f) 75 and (g) 100 pM.

The specificity of the proposed method for FR detection was investigated by comparing the color change of the probe solution with the presence of the target FR against other control proteins, including BSA, SA, and IgG. Based on the results shown in Fig. 4, the presence of the control proteins (each at the concentration of 50 pM) causes negligible color changes compared with the blank test, while the addition of the target FR (50 pM) leads to clear color change, indicating the high selectivity of the method. This further reveals that the control proteins are unable to interact with FA-ssDNA and to prevent FA-ssDNA from digesting by Exo I, leading to negligible color changes of the probe solutions.

Fig. 4 Selectivity investigation of the proposed colorimetric method for the detection of FR against other control proteins, (a) blank, (b) BSA, (c) SA, (d) IgG and (e) FR all at identical concentrations of 50 pM.

To check the potential applicability of the proposed method for the monitoring of FR in real samples, recovery tests for FR in diluted human serum samples (10%) with the standard addition approach were performed. According to the results list in Table 1, the recoveries for the added FR fall in the range from 94% to 103%, indicating that the developed method is suitable for real samples.

Table 1 Analysis of FR in diluted human serum samples

Samples	Added FR (pM)	Found FR (mean ± RSD, pM)	Recovery (%)
1	5	4.7 ± 0.61	94
2	20	19.5 ± 1.6	97.5
3	50	50.9 ± 1.9	101
4	75	77.5 ± 3.5	103

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In summary, we have shown that the coupling of terminal protection of FA-ssDNA with HCR amplification formation of the DNAzyme polymers can lead to label-free and highly sensitive colorimetric detection of FR. The association of FR with FA-ssDNA protects FA-ssDNA from digesting by Exo I, and the protected ssDNA further triggers the self-assembly of G-quadruplex sequence-containing hairpin DNAs to form DNAzyme polymers, which cause significantly intensified color change of the probe solution to achieve highly sensitive detection of FR down to 5 pM with naked eye. Besides, without the involvement complex instruments, our approach for protein biomarker detection is cost-effective. Moreover, the developed sensing method is also selective against the control proteins. With these advantages, the reported approach can be easily expanded for label-free and sensitive detection of different types of small molecule/protein interactions by changing the corresponding affinity pairs (e.g., biotin/streptavidin), which provides new alternatives for early diagnosis of cancers and drug developments.

Acknowledgements

This work was supported by NSFC (nos 21275004 and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932) and Fundamental Research Funds for the Central Universities (XDJK2014A012).

Notes and references

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