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Article

Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis

by 1, 1,2,* and 1,2,*
1
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
2
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266235, China
*
Authors to whom correspondence should be addressed.
Academic Editor: Sean M. Kerwin
Molecules 2022, 27(12), 3706; https://doi.org/10.3390/molecules27123706
Received: 15 April 2022 / Revised: 27 May 2022 / Accepted: 6 June 2022 / Published: 9 June 2022
(This article belongs to the Special Issue Z-DNA and Z-RNA: from Physical Structure to Biological Function)
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. View Full-Text
Keywords: Z-DNA; binding constant; ZBP1; circular DNA; bio-layer interferometry Z-DNA; binding constant; ZBP1; circular DNA; bio-layer interferometry
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MDPI and ACS Style

Li, L.; An, R.; Liang, X. Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis. Molecules 2022, 27, 3706. https://doi.org/10.3390/molecules27123706

AMA Style

Li L, An R, Liang X. Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis. Molecules. 2022; 27(12):3706. https://doi.org/10.3390/molecules27123706

Chicago/Turabian Style

Li, Lin, Ran An, and Xingguo Liang. 2022. "Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis" Molecules 27, no. 12: 3706. https://doi.org/10.3390/molecules27123706

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