{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Ramirez Montero D"],"funding":["European Molecular Biology Organization","Boehringer Ingelheim Foundation","European Research Council","Dutch Research Council (NWO)"],"pagination":["31-41"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC10808024"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["123(1)"],"pubmed_abstract":["DNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/noncanonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/noncanonical structures into plasmids often requires several rounds of cloning on pre-existing plasmids whose sequence one cannot fully control. Here, we describe a simple and efficient way to synthesize 10.1-kb plasmids de novo using synthetic gBlocks that provides full control of the sequence. Using these plasmids, we developed a 1.5-day protocol to assemble 10.1-kb linear DNA constructs with end and internal modifications. As a proof of principle, we synthesize two different DNA constructs with biotinylated ends and one or two internal 3' single-stranded DNA flaps, characterize them using single-molecule force and fluorescence spectroscopy, and functionally validate them by showing that the eukaryotic replicative helicase Cdc45/Mcm2-7/GINS (CMG) binds the 3' single-stranded DNA flap and translocates in the expected direction. We anticipate that our approach can be used to synthesize custom-sequence DNA constructs for a variety of force and fluorescence single-molecule spectroscopy experiments to interrogate DNA replication, DNA repair, and transcription."],"journal":["Biophysical journal"],"pubmed_title":["De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features."],"pmcid":["PMC10808024"],"funding_grant_id":["789267","714.017.002"],"pubmed_authors":["Ramirez Montero D","Liu Z","Dekker NH"],"additional_accession":[]},"is_claimable":false,"name":"De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features.","description":"DNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/noncanonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/noncanonical structures into plasmids often requires several rounds of cloning on pre-existing plasmids whose sequence one cannot fully control. Here, we describe a simple and efficient way to synthesize 10.1-kb plasmids de novo using synthetic gBlocks that provides full control of the sequence. Using these plasmids, we developed a 1.5-day protocol to assemble 10.1-kb linear DNA constructs with end and internal modifications. As a proof of principle, we synthesize two different DNA constructs with biotinylated ends and one or two internal 3' single-stranded DNA flaps, characterize them using single-molecule force and fluorescence spectroscopy, and functionally validate them by showing that the eukaryotic replicative helicase Cdc45/Mcm2-7/GINS (CMG) binds the 3' single-stranded DNA flap and translocates in the expected direction. We anticipate that our approach can be used to synthesize custom-sequence DNA constructs for a variety of force and fluorescence single-molecule spectroscopy experiments to interrogate DNA replication, DNA repair, and transcription.","dates":{"release":"2024-01-01T00:00:00Z","publication":"2024 Jan","modification":"2026-05-29T11:38:06.572Z","creation":"2025-04-05T16:59:12.167Z"},"accession":"S-EPMC10808024","cross_references":{"pubmed":["37968907"],"doi":["10.1016/j.bpj.2023.11.008"]}}