Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis
The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) w...
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| Format: | Article |
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MDPI AG
2022-06-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/27/12/3706 |
| _version_ | 1797484094165090304 |
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| author | Lin Li Ran An Xingguo Liang |
| author_facet | Lin Li Ran An Xingguo Liang |
| author_sort | Lin Li |
| collection | DOAJ |
| description | The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5′-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules. |
| first_indexed | 2024-03-09T22:56:34Z |
| format | Article |
| id | doaj.art-d2891a58e4d24048a1fab56202826b25 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T22:56:34Z |
| publishDate | 2022-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-d2891a58e4d24048a1fab56202826b252023-11-23T18:09:52ZengMDPI AGMolecules1420-30492022-06-012712370610.3390/molecules27123706Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding AnalysisLin Li0Ran An1Xingguo Liang2College of Food Science and Engineering, Ocean University of China, Qingdao 266003, ChinaCollege of Food Science and Engineering, Ocean University of China, Qingdao 266003, ChinaCollege of Food Science and Engineering, Ocean University of China, Qingdao 266003, ChinaThe binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5′-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules.https://www.mdpi.com/1420-3049/27/12/3706Z-DNAbinding constantZBP1circular DNAbio-layer interferometry |
| spellingShingle | Lin Li Ran An Xingguo Liang Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis Molecules Z-DNA binding constant ZBP1 circular DNA bio-layer interferometry |
| title | Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis |
| title_full | Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis |
| title_fullStr | Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis |
| title_full_unstemmed | Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis |
| title_short | Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis |
| title_sort | construction of ssdna attached lr chimera involving z dna for zbp1 binding analysis |
| topic | Z-DNA binding constant ZBP1 circular DNA bio-layer interferometry |
| url | https://www.mdpi.com/1420-3049/27/12/3706 |
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