Efficient modification of λ-DNA substrates for single-molecule studies

Abstract Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifi...

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Autores principales:	Yoori Kim, Armando de la Torre, Andrew A. Leal, Ilya J. Finkelstein
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8b70cb40cf1a47ca9a485db753930e3d
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spelling	oai:doaj.org-article:8b70cb40cf1a47ca9a485db753930e3d2021-12-02T16:07:48ZEfficient modification of λ-DNA substrates for single-molecule studies10.1038/s41598-017-01984-x2045-2322https://doaj.org/article/8b70cb40cf1a47ca9a485db753930e3d2017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01984-xhttps://doaj.org/toc/2045-2322Abstract Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90–100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5′-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.Yoori KimArmando de la TorreAndrew A. LealIlya J. FinkelsteinNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yoori Kim Armando de la Torre Andrew A. Leal Ilya J. Finkelstein Efficient modification of λ-DNA substrates for single-molecule studies
description	Abstract Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90–100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5′-ssDNA flaps, (CAG)13 triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)13 structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.
format	article
author	Yoori Kim Armando de la Torre Andrew A. Leal Ilya J. Finkelstein
author_facet	Yoori Kim Armando de la Torre Andrew A. Leal Ilya J. Finkelstein
author_sort	Yoori Kim
title	Efficient modification of λ-DNA substrates for single-molecule studies
title_short	Efficient modification of λ-DNA substrates for single-molecule studies
title_full	Efficient modification of λ-DNA substrates for single-molecule studies
title_fullStr	Efficient modification of λ-DNA substrates for single-molecule studies
title_full_unstemmed	Efficient modification of λ-DNA substrates for single-molecule studies
title_sort	efficient modification of λ-dna substrates for single-molecule studies
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/8b70cb40cf1a47ca9a485db753930e3d
work_keys_str_mv	AT yoorikim efficientmodificationofldnasubstratesforsinglemoleculestudies AT armandodelatorre efficientmodificationofldnasubstratesforsinglemoleculestudies AT andrewaleal efficientmodificationofldnasubstratesforsinglemoleculestudies AT ilyajfinkelstein efficientmodificationofldnasubstratesforsinglemoleculestudies
_version_	1718384728646615040

Efficient modification of λ-DNA substrates for single-molecule studies

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