Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies
Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require dist...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/19/11404 |
| _version_ | 1797479048025210880 |
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| author | Zhiqiang Sun Tommy Stormberg Shaun Filliaux Yuri L. Lyubchenko |
| author_facet | Zhiqiang Sun Tommy Stormberg Shaun Filliaux Yuri L. Lyubchenko |
| author_sort | Zhiqiang Sun |
| collection | DOAJ |
| description | Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes. |
| first_indexed | 2024-03-09T21:40:18Z |
| format | Article |
| id | doaj.art-8b8f78c3cf7f440588cf2cb7f6fa8c9e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T21:40:18Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-8b8f78c3cf7f440588cf2cb7f6fa8c9e2023-11-23T20:32:36ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-09-0123191140410.3390/ijms231911404Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy StudiesZhiqiang Sun0Tommy Stormberg1Shaun Filliaux2Yuri L. Lyubchenko3Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USADepartment of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USADepartment of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USADepartment of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USAAtomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes.https://www.mdpi.com/1422-0067/23/19/11404Atomic Force Microscopy (AFM)three-way junction (3WJ)DNA labelnucleosomes |
| spellingShingle | Zhiqiang Sun Tommy Stormberg Shaun Filliaux Yuri L. Lyubchenko Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies International Journal of Molecular Sciences Atomic Force Microscopy (AFM) three-way junction (3WJ) DNA label nucleosomes |
| title | Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies |
| title_full | Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies |
| title_fullStr | Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies |
| title_full_unstemmed | Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies |
| title_short | Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies |
| title_sort | three way dna junction as an end label for dna in atomic force microscopy studies |
| topic | Atomic Force Microscopy (AFM) three-way junction (3WJ) DNA label nucleosomes |
| url | https://www.mdpi.com/1422-0067/23/19/11404 |
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