Sen Lia,
Yu Lia,
Hongwei Yua,
Zhan Wu*b,
Jianhui Jiangb,
Ruqin Yub and
Yuansheng Wang*a
aCollege of Science, The Naval University of Engineering, Wuhan, 430000, P. R. China. E-mail: qianxun8965@163.com; Fax: +86-027-68771774; Tel: +86-027-68771774
bState Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China. E-mail: zhanwu@hnu.edu.cn; Fax: +86-731-8822782; Tel: +86-731-8822577
First published on 14th December 2015
A novel fluorescence assay strategy has been developed for quantitatively detecting small molecule–protein interactions on the basis of terminal protection. It is well-known that β-indole acetic acid (IAA) is a critical plant hormone molecule regulating plant growth. This terminal protection strategy was demonstrated using IAA and its binding protein anti-IAA antibody as a model case. The IAA-labeled strand of the double-stranded DNA (dsDNA) is protected from degradation by T7 exonuclease when the small molecule moiety is bound to its antibody. By using a competitive assay format, this developed method shows that the fluorescence peaks are dynamically correlated to the concentrations of IAA ranging from 1 to 2000 nM with a detection limit of 0.4 nM. Our assay strategy is able to offer high selectivity, excellent reproducibility, cost-effective, and simplified operations.
However, many of the existing methods require substantially improved throughput and decreased time, cost, and material consumption (Table S1 in ESI†). Thus, a universal protocol for cost-effective, highly sensitive, simplified operation and selective detection of small molecules remains a major opportunity and challenge.
It is well-known that IAA is a critical plant hormone molecule regulating the plant growth. Although several assay strategies have been applied in IAA detection,10 such as electrochemistry and fluorescence resonance energy transfer,11 no fluorescence amplification assay strategy using small molecule–protein interaction via terminal protection has been reported as far as we know.12 Considering the poor sensitivities of current analytical techniques, an effective and sensitive analytical protocol for the detection of IAA is especially important to the agriculture and other related fields.
Jiang group has reported on a terminal protection strategy that is conducted by the specific binding of small molecule-labeled DNA and target protein.13 Oligonucleotide probes tethered with specific target molecules provides with the versatile capacity to specifically bind to the target protein. The double-stranded DNA hybrid offers the capacity of signal transduction and amplification.14
This assay is based on our reasoning that the small molecule-linked strand of the double-stranded DNA (dsDNA) is protected from the degradation by T7 exonuclease when the small molecule moiety is bound to its protein target.15 This terminal protection strategy was demonstrated using β-indole acetic acid (IAA) and its binding protein anti-IAA antibody as a model case. T7 exonuclease has been reported to degrade the double-stranded DNA (dsDNA) in the 5′ to 3′ direction.16 Probe 2 with a 5-carboxy fluorescein (FAM) tag at the 5′ end and a BHQ-1 quencher in adjacent nucleotides is designed to perfectly complement the small molecule-linked DNA (probe 1).
The interaction of small molecule-linked DNA (probe 1) with its protein may dramatically increases steric hindrance, which protects the FAM-labeled probe (probe 2)/probe 1 hybrid from T7 exonuclease-catalyzed digestion. This strategy is essentially a competitive assay format between the labeled and free small molecules, which compete for binding the antibody protein.
In the present of free IAA, the free IAA molecules are bound to the anti-IAA antibodies by preincubating in the target sample solution. Thus, the IAA-labeled DNA (probe 1) cannot interact with anti-IAA antibody. Without the terminal protection of the small molecule–protein interactions, T7 exonuclease cyclically cleaves the fluorescence-quenched DNA probe successively into mononucleotides from the 5′ terminal, thereby activating an obviously strong fluorescence signal. Namely, the displaced labeled-probe become available for binding another FAM-labeled probe (probe 2) and triggering new amplification cycles.
Because cyclical cleavage of the input the FAM-labeled probe affords efficient amplification of the fluorescence signal, this strategy is able to ensure substantial signal amplification and a low background current. By using a competitive assay format between the labeled IAA and the free IAA, this developed method shows that the fluorescence emission peaks are dynamically correlated to the concentrations of IAA range from 1 to 2000 nM with a detection limit of 0.4 nM. Such a developed assay technology offers outstanding advantages over existing techniques such as the surface-based methods. Thus, this assay strategy may offer a simple, highly selective, excellent reproducibility, cost-effective, and simplified detection platform for identification and quantification of small molecule–protein interactions.
The conjugate was dialyzed against 10 mM PBS (1000 mL) for 24 h to remove excess by using a membrane with molecular weight cutoff of 1000 Da. The dialysis was performed for 3 days with shielding from light and changes with fresh buffer every 4–5 h.
The principle mechanism of terminal protection assay is that binding a protein to small molecule-labeled DNA will may dramatically increases steric hindrance around the binding site, and thus prevent the FAM-labeled probe (probe 2)/probe 1 hybrid from T7 exonuclease-catalyzed digestion. As for the reaction mixture containing target IAA molecules, the free IAA molecules are bound to the anti-IAA antibodies by preincubating in the target sample solution. Thus, the IAA-labeled DNA (probe 1) cannot interact with anti-IAA antibody. Without the terminal protection of the small molecule–protein interactions, T7 exonuclease cyclically cleaves the fluorescence-quenched DNA probe successively into mononucleotides from the 5′ terminal, thereby a significantly strong fluorescence signal was obtained. Namely, the displaced labeled-probe was released to bind another FAM-labeled probe (probe 2) and rendering a new amplification cycle. Due to cyclical cleavage of the input FAM-labeled probe, this efficient fluorescence signal amplification strategy may have a great potential for the detection of small molecule–protein interactions.
It was verified that the terminal protection mechanism could work with T7 exonuclease. To verify terminal protection of IAA-labeled DNA probe 1 and 2 against T7 exonuclease, we design a gel electrophoresis assay strategy. T7 exonuclease has been reported to degrade the double-stranded DNA (dsDNA) in the 5′ to 3′ direction. The interaction of small molecule-linked DNA (probe 1) with its protein may effectively increases steric hindrance, which protects the FAM-labeled probe (probe 2)/probe 1 hybrid from the digestion of T7 exonuclease.
As shown in Fig. 1, the DNA double-strand hybrid duplex generated by the hybridization of unlabeled DNA probe 1 with the probe 2 (lane 1), the double-stranded DNA duplex generated by the hybridization of IAA-labeled DNA probe 1 with the probe 2 (lane 2) exhibited two bright bands with the same mobility.
After adding T7 exonuclease into the reaction mixture above, it was observed that almost no bright band (lane 4) compared with the bright band in lane 3. Because of the degraded DNA mononucleotides would migrate out of the gel and only double-strand DNA oligonucleotides could be stained selectively using ethidium bromide, we could infer that probe 2 was digested by T7 exonuclease into mononucleotides without the terminal protection of IAA molecule–antibody interaction. Thus, the appearance of visible bands in the gel could directly verify the terminal protection of anti-IAA antibody and the labeled IAA could inhibit the action of T7 exonuclease. Considering the existing methods require substantially improved throughput and decreased time, it is of tremendous importance to develop an assay strategy for small molecule detection based on signal transduction and amplification protocol. On the basis of the terminal protection, this sensitive and selective fluorescence assay strategy may hold considerable potential in small molecule–protein interaction investigations.
As shown in Fig. 2, the terminal protection of IAA-labeled DNA probe 1 and 2 against T7 exonuclease was further studied using fluorescence analysis. Each sample was incubated with T7 exonuclease at 25 °C for 100 min before the fluorescence measurement. Without the addition of the anti-IAA antibody, the digested probe 2 separates the fluorophore from the quencher, activates the fluorescence signal. The end-point fluorescence readout showed a dramatically higher fluorescent intensity, compared with the presence of anti-IAA antibody. These datas demonstrated that binding of anti-IAA antibody to the IAA label could prevent the IAA-labeled DNA probe 1/probe 2 hybrid from cleaving.
![]() | ||
Fig. 2 Fluorescence intensity in absence (red) and the presence (black) of the anti-IAA antibody. The excitation wavelength was 494 nm and the emission wavelength was from 505 nm to 600 nm. |
As shown in Fig. 3, we could clearly see the time-dependent fluorescence responses under different conditions. Each sample was incubated at 37 °C for 45 min before the real-time fluorescence measurement. From Fig. 3A, in the mixture containing only 100 nM unlabeled DNA probe, 500 nM probe 2, 100 μg mL−1 anti-IAA, a very weak fluorescence signal (∼201), was observed after an incubation of 2 h at 25 °C, and the fluorescence readout did not show appreciable time-dependent changes. When adding T7 exonuclease into the systems containing 100 nM unlabeled DNA probe, 500 nM probe 2, 100 μg mL−1 anti-IAA and 1 U μL−1 T7 exonuclease, the fluorescence was remarkably enhanced, and confirming that probe 2 was digested by T7 exonuclease. From Fig. 3B, in the presence of anti-IAA antibody, the mixture showed a much slower rate of fluorescence activation. After an incubation at 25 °C for 2 h, the end-point fluorescence readout showed only a ∼3-fold enhancement, compared with that for the noncleaved probe 2 (line b). We could observed that, in the absence of IAA, this system gave a high signal-to-background ratio compared with the fluorescence intensity for the addition of anti-IAA antibody. This immediate evidence confirmed that the binding of anti-IAA antibody to the IAA label could prevent the IAA-labeled DNA probe 1/probe 2 hybrid from cleaving.
Furthermore, as shown in Fig. 3C, we replaced the anti-IAA by another protein BSA in the system to test terminal protection of the labeled-IAA and the anti-IAA antibody. We then performed further experiment under the condition of excessive free IAA, it was observed that, the system gave almost the same fluorescence intensity for the mixture of IAA-labeled DNA probe 1, BSA, probe 2 and T7 exonuclease. These results indicated that inhibition of T7 exonuclease digestion activity of the system specifically arises from the binding of anti-IAA antibody to the IAA label.
Fig. 4A shows typical the fluorescence peaks of terminal protection assay in response to IAA of varying concentrations. The fluorescence responses of IAA-labeled biosensor in competitive assay of the free IAA were observed, and the fluorescence peaks were dynamically increased with increasing concentrations of IAA within the range of 1 to 2000 nM.
![]() | ||
Fig. 4 (A) Fluorescence responses of IAA-labeled biosensor in competitive assay of the free IAA. (B) Fluorescence intensity at 520 nm versus the concentration of the free IAA. |
As shown in Fig. 4B, the fluorescence peak intensity versus IAA concentrations showed a linear correlation ranging from 0 to 20 nM. The detection limit was estimated to be 0.4 nM according to the 3σ rule. Relative standard deviations (RSDs) of fluorescence intensity at 520 nm were 3.09%, 3.35%, 2.77%, and 2.85% in four repetitive assays of 20, 200, 500, and 2000 nM IAA. Also, the developed strategy exhibited a very desirable reproducibility, high signal-to-background ratio and a wide dynamic response range based on its fluorescence amplification detection assay and competitive assay format for the detection of small molecule–protein interactions.
Specificity evaluation of terminal protection assay strategy for IAA detection was shown in Fig. 5. It was observed that the fluorescence spectrometer yielded very weak fluorescence peaks in response to these plant hormone small molecules such as GA, CTK, ABA, which contrasted clearly with the strong fluorescence response to IAA.
In the specificity evaluation using the assay system with different plant hormone small molecules, we obtained very small fluorescence peaks, implying that fluorescence assay strategy was excellent selective.
Footnote |
† Electronic supplementary information (ESI) available: DNA sequences and additional figures. See DOI: 10.1039/c5ra20240j |
This journal is © The Royal Society of Chemistry 2015 |