Towards development of the 4c-based method detecting interactions of plasmid dna with host genome

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Abstract

Chromosome conformation capture techniques have revolutionized our understanding of chromatin architecture and dynamics at the genome-wide scale. In recent years, these methods have been applied to a diverse array of species, revealing fundamental principles of chromosomal organization. However, structural organization of the extrachromosomal entities, like viral genomes or plasmids, and their interactions with the host genome, remain relatively underexplored. In this work, we introduce an enhanced 4C-protocol tailored for probing plasmid DNA interactions. We design specific plasmid vector and optimize protocol to allow high detection rate of contacts between the plasmid and host DNA.

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About the authors

A. P. Yan

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State University

Author for correspondence.
Email: a.yan@g.nsu.ru
Russian Federation, Novosibirsk; Novosibirsk

P. A. Salnikov

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State University

Email: a.yan@g.nsu.ru
Russian Federation, Novosibirsk; Novosibirsk

M. M. Gridina

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State University

Email: a.yan@g.nsu.ru
Russian Federation, Novosibirsk; Novosibirsk

P. S. Belokopytova

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State University

Email: a.yan@g.nsu.ru
Russian Federation, Novosibirsk; Novosibirsk

V. S. Fishman

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences; Novosibirsk State University

Email: a.yan@g.nsu.ru
Russian Federation, Novosibirsk; Novosibirsk

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2. Fig. 1. Design of the 4C experiment with a plasmid vector. a – General design of the experiment (I–VI). b – Scheme of the arrangement of restriction sites of the plasmid vectors used in this work. The maps and sequences of the plasmids are presented in Fig. A1–A3 and in Text 2 in Appendix

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3. Fig. 2. Estimation of the proportion of “random” contacts. a – Experimental scheme: plasmid #1 was transfected into HEK293T cells (I), then spatial contacts of DNA and proteins in the nucleus were fixed with formaldehyde (II). Fixed Mus musculus fibroblasts (III), plasmid #2 (IV) were added to the resulting sample and a 4C experiment was performed. b – Plasmid #1 and plasmid #2 differ in the ratio of the number of contacts with the chromatin of H. sapiens and M. musculus. The diagrams show the percentage of plasmid contacts with the corresponding chromatin, averaged over three independent experiments. The results for individual replicates are presented in Fig. A4 in the Appendix. The total number of contacts is taken as 100%.

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