A dsDNA-lighted fluorophore for monitoring protein-ligand interaction through binding-mediated DNA protection

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SCIENCE CHINA Chemistry
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SCIENCE CHINA Chemistry, Volume 61, Issue 12: 1630-1636(2018) https://doi.org/10.1007/s11426-018-9349-9

A dsDNA-lighted fluorophore for monitoring protein-ligand interaction through binding-mediated DNA protection

Zhihe Qing*, Lixuan Zhu, Lina Hou, Zhen Zou, Sheng Yang, Ronghua Yang*
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  • ReceivedApr 16, 2018
  • AcceptedAug 22, 2018
  • PublishedOct 30, 2018
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Abstract

Because of their important roles in cellular functions, life activities, drug screening, and disease treatment, the development of efficient methods for monitoring protein-ligand interaction is essential. In this study, inspired by our previous studies on DNA conformation-selective fluorescent indicators, we developed a new sensing platform for monitoring protein-ligand interaction and detecting protein activity based on binding-mediated DNA protection and the dsDNA-lighted fluorophore, ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] butanoate (EBCB). The ligand was purposefully linked to the 3?-terminal of a hairpin DNA probe to selectively bind with the target protein and protect the DNA from cleavage by exonuclease III. By virtue of EBCB’s outstanding capacity to discriminate DNA conformation, the protein-ligand interaction could be effectively monitored through a fluorescence change in EBCB. A high fluorescence signal was detected when the hairpin DNA was protected in the presence of the target protein, whereas a much lower signal was observed in the presence of nontarget proteins. Our results demonstrated that the proposed strategy had high potential, such as high selectivity and relatively high sensitivity, for monitoring protein-ligand interaction and detecting protein activity. We believe these results will pave the way for applying dsDNA-lighted fluorophore EBCB as an effective signal transducer for DNA conformation transformation-mediated biochemical sensing.


Funded by

the National Natural Science Foundation of China(21605008,21735001,21575018,21505006)

the Hunan Provincial Natural Science Foundation(2016JJ3001)


Acknowledgment

This work was supported by the National Natural Science Foundation of China (21605008, 21735001, 21575018, 21505006) and the Hunan Provincial Natural Science Foundation (2016JJ3001).


Interest statement

The authors declare that they have no conflict of interest.


Supplement

Supporting Information

The supporting information is available online at http://chem.scichina.com and http://link.springer.com/journal/11426. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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    Scheme 1

    Schematic illustration of monitoring protein-ligand interaction and protein activity based on EBCB and recognition-mediated transformation of DNA conformation (color online).

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    Figure 1

    The synthetic route of ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] butanoate (color online).

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    Figure 2

    (a) Fluorescence spectra of EBCB responding to 250 nM ssDNA and dsDNA. DNA1 was used as ssDNA, dsDNA was the hybridization product of DNA1 and DNA2, and the detailed sequence information is listed in Table S1. (b) Typical fluorescence responses of EBCB to the hairpin structural DNA (HP1) probe and other components (color online).

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    Figure 3

    Investigation of the selectivity of protein-ligand interaction. (a) Fluorescence spectra of EBCB responding to different proteins when the biotin-labeled hairpin DNA (HP1) probe was used. (b) Fluorescence spectra of EBCB responding to different proteins when unlabeled hairpin DNA (HP2) was used. The concentration of streptavidin (SA) was 100 nM, and other proteins were at 200 nM (color online).

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    Figure 4

    (a) Fluorescence spectra of HP1/EBCB system in the presence of target streptavidin (SA) at different concentrations. EBCB fluorescence gradually increased with the increasing of target concentration from 0 to 150 nM. (b) The relationship between the fluorescence intensity of EBCB and SA concentration. Inset shows the linear range from 2 to 80 nM (color online).

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    Figure 5

    Monitoring protein denaturation as a function of temperature. (a) Fluorescence spectra of HP1/EBCB system responding to streptavidin (SA) pretreated under different temperatures; (b) relative activity of SA after treatment at different temperatures; the result from 30?°C treatment was normalized to 100%. Relative activity was defined as [(FT--F without SA)/(F30--F without SA)]×100%, where F30 is the fluorescence intensity of HP1/EBCB when SA was pretreated at 30?°C, F without SA was that of the blank with SA, FT was that when SA was pretreated at a specific temperature (color online).

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