High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging

Gallardo, Iluminada; Pasupathy, Praveenkumar; Brown, Maxwell W.; Manhart, Carol M.; Neikirk, Dean P.; Alani, Eric; Finkelstein, Ilya J.

doi:10.1021/acs.langmuir.5b02416

Cited by 58 publications

(74 citation statements)

References 78 publications

(156 reference statements)

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“…In this assay, a fluid lipid bilayer is deposited on the surface of a micro-patterned flowcell 32–34 . Biotinylated DNA is immobilized on the lipid bilayer via a biotin-streptavidin linkage and buffer flow is used to organize hundreds of DNA molecules at chromium (Cr) diffusion barriers (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Kim

Torre

Leal

et al. 2017

Sci Rep

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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.

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Section: Resultsmentioning

confidence: 99%

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

Kim

Torre

Leal

et al. 2017

Sci Rep

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“…3A, B)(Myler et al, 2016). In this assay, thousands of individual DNA molecules are organized at fabricated structures in a microfluidic flowcell for concurrent single-molecule imaging (Gallardo et al, 2015). To assemble DNA curtains, a supported lipid bilayer is first deposited on the surface of a microfluidic flowcell.…”

Section: Long-range Resectionmentioning

confidence: 99%

Eukaryotic resectosomes: A single-molecule perspective

Myler

Finkelstein

2017

Progress in Biophysics and Molecular Biology

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DNA double-strand breaks (DSBs) disrupt the physical and genetic continuity of the genome. If unrepaired, DSBs can lead to cellular dysfunction and malignant transformation. Homologous recombination (HR) is a universally conserved DSB repair mechanism that employs the information in a sister chromatid to catalyze error-free DSB repair. To initiate HR, cells assemble the resectosome: a multi-protein complex composed of helicases, nucleases, and regulatory proteins. The resectosome nucleolytically degrades (resects) the free DNA ends for downstream homologous recombination. Several decades of intense research have identified the core resectosome components in eukaryotes, archaea, and bacteria. More recently, these proteins have been characterized via single-molecule approaches. Here, we focus on recent single-molecule studies that have begun to unravel how nucleases, helicases, processivity factors, and other regulatory proteins dictate the extent and efficiency of DNA resection in eukaryotic cells. We conclude with a discussion of outstanding questions that can be addressed via single-molecule approaches.

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“…To assemble DNA curtains, DNA molecules are anchored to a supported lipid bilayer (SLB) via a biotin–streptavidin linkage and organized at patterned features on the flowcell surface (Finkelstein & Greene, 2011; Gallardo et al, 2015). SLBs offer three key advantages for single-molecule studies of protein–DNA interactions.…”

Section: Methodsmentioning

confidence: 99%

“…This protocol describes a rapid UV lithography-based process for fabricating quartz slides for DNA curtains imaging (Gallardo et al, 2015) (summarized in Fig. 2).…”

Section: Methodsmentioning

confidence: 99%

Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

Soniat

Myler

Schaub

et al. 2017

Methods in Enzymology

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Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.

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High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging

Cited by 58 publications

References 78 publications

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

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

Eukaryotic resectosomes: A single-molecule perspective

Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

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