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DNA curtain

From EverybodyWiki Bios & Wiki



DNA curtain is a single-molecule imaging technique used to visualize protein–DNA interactions in real time with high-throughput. This method arranges long double-stranded or single-stranded DNA molecules on a supported lipid bilayer within a microfluidic chamber, where hydrodynamic flow aligns them against nanofabricated diffusion barriers. Traditional single-molecule techniques typically observe only one molecule at a time. These approaches can be limited by low statistical relevance or nonspecific surface adsorption.[1] In contrast, DNA curtains allow hundreds to thousands of DNA molecules to be monitored simultaneously using total internal reflection fluorescence microscope (TIRFM). This high-throughput capability enables direct observation of how proteins interact with DNA, translocate along it, and search for target sequences.

History

The DNA curtains technique was first introduced by Eric C. Greene and colleagues at Columbia University. Early methods used supported lipid bilayers and nanofabricated diffusion barriers to align long DNA molecules into parallel arrays for high-throughput single-molecule imaging.[2] Subsequent work incorporated nanoscale diffusion barriers (“curtain rods”) fabricated using electron-beam and nanoimprint lithography, which provided more precise control over the organization and orientation of DNA molecules on the surface.[3] Greene’s group later standardized DNA curtain protocols for total internal reflection fluorescence microscopy and adopted chromium-based diffusion barriers patterned on fused silica substrates.[3] During this period, double-tethered DNA curtains were developed, allowing DNA molecules to remain extended without continuous buffer flow. In 2012, single-stranded DNA curtains were introduced by Gibb, Silverstein, Finkelstein, and Greene, extending the technique to ssDNA substrates.[4]

In 2015, Gallardo at Cornell University introduced a photolithography-based method for large-scale fabrication of chromium diffusion barriers, allowing the assembly of hundreds of DNA arrays within a single flow cell for high-throughput single-molecule studies.[5] In 2021, Kopuštas and co-workers introduced Soft DNA Curtains, a lithography-free method that uses microcontact-printed protein templates to orient and immobilize DNA molecules with single or double end tethering. This approach enhances stability and enables high-throughput single-molecule studies of protein–DNA interactions.[6] DNA curtains have continuously evolved through improvements in nanofabrication, surface patterning, and tethering strategies. The technique has become a widely used high-throughput platform for observing DNA–protein interactions across diverse biological systems.

Principles

Flow cell assembly

Schematic of a flow cell assembly

DNA curtain experiment typically begins with the assembly of a microfluidic flow cell composed of a fused-silica slide and a borosilicate glass coverslip. A narrow channel is formed between the two surfaces using double-sided adhesive tape, which creates the internal chamber for buffer and sample exchange.[7] Inlet and outlet ports are glued over the drilled holes in the flow cell, and syringes are attached to these ports to support bilayer deposition.[8]

Once assembled, the flow cell is placed on a remotely operated microscope stage, which helps with accurate positioning during total internal reflection fluorescence microscope (TIRFM) imaging. Before use, the flow cells are connected to a syringe pump to control the buffer flow through the chamber and an HPLC six-way injection valve to control sample delivery.[2] This configuration creates a stable, sealed, and optically clear microfluidic environment that is well suited for supported lipid bilayer formation, DNA tethering, and regulated buffer flow.

Diffusion barriers

Diffusion barriers play an important role in the DNA curtains technique by organizing lipid-tethered DNA molecules into orderly arrays, stopping each strand at one end as it diffuses across the membrane.[9] The barrier patterns can be designed in various shapes depending on the experimental requirements. The first design, barrier 1.0,[10] relied on simple mechanical scratches in quartz to create raised ridges that halted DNA diffusion. However, the uneven features made it difficult to achieve consistent tethering positions. Barrier 2.0[3] improved positional precision using nanoscale chromium lines fabricated by electron-beam lithography and thermal evaporation. Although its straight geometry still permitted lateral DNA diffusion. To address this, barrier 3.0[11] introduced a zig-zag pattern that effectively prevents lateral diffusion and subsequently became the standard design.

A later version, barrier 4.0,[12] uses discontinuous patterns that improve the ability to distinguish individual DNA molecules, particularly at higher DNA densities. Regardless of design, the barriers function to halt the lateral diffusion of DNA within the supported lipid bilayer. Under hydrodynamic flow, tethered DNA migrates until it encounters the barrier, where it becomes trapped and extended. By controlling DNA concentration, individual molecules can be isolated within each barrier feature.[13]

Lipid bilayer formation

Supported lipid bilayers (SLBs) provide a passivated and fluid surface for organizing tethered DNA molecules. The bilayer is typically formed by adsorbing vesicles composed of dioleoylphosphatidylcholine (DOPC), biotinylated-dioleoyl phosphatidylethanolamine (DOPE), and methoxy polyethylene glycol (mPEG) DOPE onto the glass flow cell surface.[7] It creates a membrane-like layer that minimizes nonspecific adsorption of proteins and nucleic acids.

Biotinylated DNA is attached to the SLB through biotin–streptavidin binding and can diffuse laterally within the two-dimensional lipid layer while remaining close to the surface for imaging.[9][14] Under hydrodynamic flow, the tethered DNA moves across the bilayer until it reaches a diffusion barrier, where it becomes aligned and extended.[14] SLBs provide strong surface passivation, reducing nonspecific binding of proteins and nucleic acids. They also allow functional lipids such as biotin or polyethylene glycol to be easily incorporated during preparation. In addition, the bilayer forms a fluid, two-dimensional surface that can be manipulated using shear or electrophoretic forces.[15] These properties make SLBs a versatile and effective platform for organizing DNA molecules in real-time single-molecule experiments.[15]

DNA tethering

DNA molecules are anchored to a supported lipid bilayer for controlled imaging and observation. In DNA curtains, both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) can be used.[9] Lambda phage DNA (48,502 bp) is commonly used as a dsDNA substrate[7] because it is commercially available and contains regions with varying adenine-thymine (AT) content, allowing studies of sequence-dependent protein binding.[9] The DNA has 12-nucleotide single-stranded overhangs, called cos ends, which can be annealed to biotin or DIG-labeled oligonucleotides for attachment. For ssDNA curtains, long single-stranded molecules are produced via rolling circle replication[4] in which a biotinylated primer anneals to a circular M13 template and is extended by Φ29 DNA polymerase.[9] Tethered DNA remains near the surface and diffuses within the lipid bilayer, facilitating alignment at diffusion barriers. This enables real-time observation of protein–DNA interactions.

Visualization

Visualization of proteins in DNA curtains experiments is achieved by labeling them with an inorganic dye,[16] attaching quantum dots,[17] or creating a fusion protein that links the protein of interest to a fluorescent tag.[18] Fusion proteins are commonly preferred because their activity can be tested to confirm that the fluorescent tag does not alter the protein’s functional properties.[13] DNA curtains containing dsDNA are visualized using intercalating dyes such as YOYO-1, while curtains containing ssDNA are typically visualized using replication protein A–eGFP.[1]

Once the DNA curtains are formed, the labeled proteins are introduced into the microfluidic flow cell and both proteins and DNA molecules are simultaneously imaged with TIRFM. The resulting image sequences are often examined using kymographs, which plot position along the DNA over time.[9] This method allows for direct, time-resolved observation of protein–DNA interactions, including diffusion, binding events, and target search processes.

Imaging

Total internal reflection fluorescence microscopy (TIRFM) is widely used in DNA curtain experiments to visualize fluorescently labeled DNA molecules and their associated proteins at single-molecule resolution. In TIRFM, a laser beam is directed through a microscope slide and reflected at an angle exceeding the critical angle, generating an evanescent electromagnetic wave at the interface between the slide and the aqueous buffer.[2] This wave penetrates a few hundred nanometers into the sample, producing a highly restricted excitation volume that significantly reduces background fluorescence compared with conventional wide-field illumination.[2]

DNA curtain assays commonly use prism-based TIRFM, in which a diode-pumped solid-state laser is focused through a fused silica prism onto a microfluidic flow cell to generate an evanescent wave.[8] The resulting fluorescence emission is collected using a high numerical aperture objective lens and passed through optical filters to remove scattered laser light. It is then detected with a back-illuminated electron-multiplying charge coupled device (EM-CCD) camera.[8] For multicolor imaging, the emitted light can be separated into distinct wavelength channels using a dichroic mirror or image-splitting device.[8] This optical configuration enables real-time imaging of nanofabricated DNA arrays and supports detailed analysis of protein–DNA interactions under physiologically relevant conditions.[2]

Procedure

In DNA curtains, the surface of a flow cell is coated with a supported lipid bilayer (SLB), with a small fraction of lipids functionalized with biotin. The bilayer is then treated with neutravidin or streptavidin, which allows biotin-labeled DNA molecules to attach.[19] Because the lipid bilayer is fluid, the DNA can move freely in two dimensions. When a hydrodynamic flow is applied, the DNA is directed toward lipid diffusion barriers on the surface. These barriers, positioned perpendicular to the flow, prevent lateral movement of the lipids, causing the DNA to accumulate along the leading edges of the barriers. Under continuous flow, hundreds or even thousands of DNA molecules align in parallel and stretch across the surface. This arrangement enables high-resolution imaging of DNA using total internal reflection fluorescence microscopy (TIRFM).[15]

Types of DNA Curtains

DNA curtains can be formed in various configurations that can be used to meet specific experimental requirements.

Conventional DNA curtains

Single-tethered DNA curtains

Schematic of a single-tethered DNA curtain

This is the earliest and simplest configuration used in DNA curtain experiments. In single-tethered curtains, each DNA molecule is attached by one end to a supported lipid bilayer through a biotin–streptavidin linkage, while the rest of the DNA extends freely into the solution.[2] When hydrodynamic buffer flow is applied, the DNA molecules are stretched and directed toward nanofabricated diffusion barriers, which align them in parallel.[20]  The resulting array of extended DNA strands can be visualized in real time using TIRFM.[21] Because each molecule is anchored only at one end, continuous flow is required to maintain extension. When the flow is stopped, the DNA recoils and diffuses away from the surface. This property provides built-in control during flow stops. Tethered DNA and any bound fluorescent proteins drift out of view, while molecules that non-specifically adsorbed to the surface remain visible.[2]

Single-tethered curtains can be created using either linear or patterned diffusion barriers. Linear barriers are simple to fabricate and effectively align DNA, but they do not regulate lateral spacing, which can lead to overlapping molecules. To achieve uniform spacing, “zigzag” barriers are used. In this design, each apex forms a small “nanowell” that captures one or more DNA molecules under flow. These zigzag barriers generate parallel lanes of separated DNA, improving single-molecule resolution in crowded fields of view.[2] The main advantages of this configuration are its simplicity and rapid assembly, making it well-suited for experiments examining binding and dissociation kinetics, such as transient protein–DNA interactions or helicase translocation under applied tension.[2][22] However, because continuous flow can influence reaction dynamics and prevent observation of freely diffusing proteins, this setup is generally used for kinetic and binding assays where DNA extension under flow is acceptable.

Double-tethered DNA Curtains

Schematic of a double-tethered DNA curtain

This configuration was developed to overcome the limitation of continuous buffer flow required in single-tethered DNA curtains. In double-tethered curtains, both ends of each DNA molecule are attached to the surface, typically one end to a supported lipid bilayer through a biotin–streptavidin linkage, and the other end is anchored to a fixed pedestal coated with anti-digoxigenin antibodies that bind to a digoxigenin (DIG) tag on the DNA.[2][17] When buffer flow is applied, the DNA is extended and aligned along nanofabricated linear or zigzag diffusion barriers, and the downstream DIG-labeled ends are captured by the pedestals. Once both ends are anchored, the DNA remains stretched and visible even when the buffer flow is stopped, allowing the entire DNA length to be imaged without hydrodynamic tension. This feature is particularly useful for experiments that require flow-free observation, such as those involving costly reagents or proteins sensitive to shear forces.[17]

Double-tethered curtains allow real-time visualization of protein–DNA interactions under near-equilibrium conditions. They provide accurate insights into diffusive motion, pausing, and enzymatic activity that could be disturbed by flow. This configuration has been widely used to study protein sliding, DNA looping, nucleosome repositioning, and condensin-mediated loop extrusion.[2][23] Overall, double-tethered DNA curtains offer greater control over molecular geometry and tension, making them one of the most versatile and informative systems for single-molecule biophysical research.

Single-stranded DNA Curtains

Schematic of a single-stranded DNA (ssDNA) curtain

ssDNA curtains are a specialized configuration of the DNA curtain platform designed for visualizing long single-stranded DNA (ssDNA) molecules at the single-molecule level. ssDNA is a key intermediate in DNA replication, homologous recombination, and DNA repair.[19] But direct imaging of long ssDNA is challenging due to its high flexibility, extensive secondary structure formation, and sensitivity to intercalating dyes, which can damage or break the ssDNA.[4] ssDNA curtains overcome these challenges by combining controlled surface tethering with fluorescent protein labeling.

Long ssDNA substrates are generated by rolling circle replication, using a circular ssDNA template, a biotinylated primer, and phi29 DNA polymerase.[19][4] The resulting ssDNA molecules are anchored to a supported lipid bilayer through biotin–streptavidin linkages, leaving the rest of each molecule free in solution.[8] When hydrodynamic buffer flow is applied, the ssDNA strands extend and align against nanofabricated diffusion barriers, forming parallel arrays suitable for real-time observation.[8] Because naked ssDNA is compact and difficult to visualize, the molecules are coated with replication protein A (RPA) fused to fluorescent proteins such as GFP or mCherry.[19] RPA binds ssDNA, removes secondary structure, and creates an extended, semi-rigid filament that can be stretched by gentle flow.[1] These RPA-ssDNA filaments are then imaged using TIRFM.

ssDNA curtains can also be assembled in a double-tethered configuration. In this setup, RPA-coated ssDNA filaments attach to anchor points downstream of the lipid diffusion barriers, providing stable extension without the need for continuous flow.[1][19] Another variation, low-complexity ssDNA curtains, uses specially designed templates that minimize base pairing, allowing individual ssDNA molecules to be synthesized and visualized in real time at the single-molecule level.[19]   ssDNA curtains have been applied to study numerous DNA-processing proteins and complexes. This method has provided key insights into the mechanisms of presynaptic complex assembly, filament remodeling, homology search, and regulation of recombination intermediates.[19]

Spatially patterned DNA curtains

Schematic of spatially patterned DNA curtains. Parallel array of double-tethered isolated DNA curtains (top), crisscrossed DNA curtains (middle), U-shaped DNA curtains (bottom).

Parallel array of double-tethered isolated DNA curtains

Parallel Array of Double-Tethered Isolated (PARDI) patterns are microfabricated structures used to produce sparsely arranged DNA curtains for single-molecule studies.[8] These patterns position double-tethered DNA molecules with a spacing of roughly 7 µm or more,[19] which minimizes interactions between neighboring strands and maintains a low local DNA concentration.[19][12] The large spacing between molecules reduces steric and kinetic interference, enabling direct observation of individual protein binding events.[8] Thus, this configuration is well suited for studying protein–DNA interactions, particularly protein association kinetics, under conditions with controlled, low DNA concentration.[8]

Crisscrossed DNA curtains  

Crisscrossed DNA curtains are a specialized arrangement of double-tethered DNA molecules designed to generate controlled regions of locally high DNA concentration.[17][19] In this configuration, individual DNA strands are organized so that they intersect at approximately 90° angles.[19] These intersections create defined crossover points where two DNA molecules come into close proximity, typically separated by ~106 nm.[17][19] Crisscrossed DNA curtains have been used to study how proteins move between two closely spaced DNA substrates, providing a controlled system for studying inter-DNA transfer and dynamic protein exchange.[17]

U-shaped DNA curtains

U-shaped DNA curtains represent a more complex configuration in which both ends of individual DNA molecules are initially immobilized on a supported lipid bilayer through biotin–streptavidin linkages.[19][24] When hydrodynamic buffer flow is applied, the DNA bends into a “U” shape, and the double-tethered molecules align along zigzag barriers that restrict lipid diffusion.[19] This arrangement allows the DNA to be visualized in real time using TIRFM. U-shaped curtains are especially useful for studying DNA compaction mechanisms, such as condensin-mediated loop extrusion, as they allow tracking of DNA length and structural changes during individual looping events with high single-molecule resolution.[19]

Advanced DNA curtains

Conventional DNA curtains rely on supported lipid bilayers and nanobarriers to organize DNA for single-molecule imaging. However, preparing lipid bilayers can be challenging, and fabricating nanobarriers is time-consuming, technically demanding, and requires specialized equipment. To overcome these limitations, several alternative configurations have been developed that retain high-throughput imaging while improving accessibility and versatility.[19]

Suspended DNA curtains

In this configuration individual DNA molecules are initially tethered to an elevated gold nanowire spanning a microfluidic flow cell through biotin–streptavidin linkages.[9][19] When buffer flow is applied, the DNA molecules are stretched along the nanowire and remain suspended above the surface, minimizing non-specific interactions with the flow cell. This arrangement enables visualization of DNA-bound proteins under lower flow rates, as the elevated nanowire creates a more uniform hydrodynamic environment.[19] Suspended DNA curtains are particularly useful for examining protein–DNA interactions in conditions that reduce surface interference. Although overlapping of nanowire-tethered DNA molecules can limit high-throughput imaging and restrict large-scale studies of these interactions.[19]

Soft DNA curtains

Soft DNA curtains are a next-generation configuration in which protein line-features are patterned on a PEGylated glass coverslip using atomic force microscopy and protein lift-off microcontact printing.[19] Streptavidin lines serve as stable anchor points for biotinylated DNA molecules, aligning them along the surface. Upon buffer flow, the DNA molecules are stretched and visualized using TIRFM.[19] Double-tethered soft DNA curtains can be formed by attaching the opposite DNA end to adjacent protein lines coated with biotinylated digoxigenin antibodies, providing defined DNA orientation.[6] This configuration enables high-throughput imaging of individual DNA molecules and facilitates studies of protein–DNA interactions, while remaining cost-effective, versatile, and accessible to users with minimal prior experience.[19]

Applications

The main application of DNA curtains is to visualize protein–DNA interactions at the single-molecule level. This method is used to study how proteins bind to DNA, search for specific targets, and move along the DNA strand.[8]

Binding site preferences

DNA curtains allow real-time visualization of protein-DNA interactions and the determination of binding site preferences. Studies showed that mismatch repair proteins, such as MutSα and MutLα, preferentially bind DNA lesions,[17] while RNA polymerase selectively occupies promoter sequences over non promoter regions.[8] This allows mapping of DNA-binding sites, tracking protein lifetimes, and observing protein–DNA interactions in real time.

Target search mechanisms

DNA curtains have been used to study how DNA binding proteins locate their target sequences.[8] By using real-time visualization of individual molecules, this method finds diffusion-based search strategies such as one-dimensional (1D) sliding, 1D hopping, intersegmental transfer, and three-dimensional (3D) diffusion.[25] Studies showed MutSα primarily uses 1D sliding,[26] while MutLα can hop and bypass nucleosomes.[17] RNA polymerase mainly relies on 3D diffusion with limited short-range facilitated diffusion.[12] This helps to understand how proteins use dynamic and mechanistic strategies to efficiently locate their targets.

Protein-protein colocalization

DNA curtains are used to study the colocalization of multiple proteins on DNA. By labeling different proteins with distinct quantum dots, it is possible to directly observe the sequential binding of proteins at specific DNA sites.[8] For example, in mismatch repair studies MutSα and MutLα are labelled with different quantum dots to monitor their interactions in real time.[17] MutSα first recognizes and binds to a DNA mismatch, after that MutLα is recruited to the same lesion.[8] This approach visualizes the formation and dynamics of protein complexes on DNA in real time.

ATP hydrolysis-driven DNA translocation

DNA curtains are used to study ATP-driven motor proteins by tracking their movement in real time. Studies used fluorescently labeled DNA translocases such as RecBCD and FtsK and tracked in real-time as they moved along the DNA substrate.[8] RecBCD rapidly travels along DNA and degrades DNA while removing other proteins from DNA,[27] whereas FtsK moves at extremely high speeds without degrading the substrate.[28] This shows how motor proteins convert chemical energy into directional movement along DNA.  

Beyond nucleic acids

DNA curtains can be used to visualize biological systems that do not involve DNA by modifying the assay to anchor proteins that regulate filament growth. For example, in actin filament formation studies,[29] formin proteins are biotinylated and attached to a supported lipid bilayer, acting as nucleation sites for filament growth.[8] When fluorescent actin monomers are introduced and a buffer flow is applied, these anchored formins guide the assembly of actin filaments along diffusion barriers, creating structured called "actin curtains."[8] This approach allows for real–time visualization of actin polymerization and provides a useful platform for studying protein–actin interactions.

Advantages

The DNA curtains method provides several advantages over traditional single-molecule techniques. Unlike most methods which observe one molecule at a time, DNA curtains enable the simultaneous imaging of hundreds or even thousands of DNA molecules, allowing high-throughput analysis. This method is specifically designed to work with long DNA strands, unlike techniques such as single-molecule FRET or pull-down methods, which typically work only with short DNA strands.[9] This allows for the study of biological processes that occur across larger regions of the genome. DNA curtains offer a practical solution to a common challenge known as the "sticking problem''.[9] By incorporating specially designed diffusion barriers, these curtains help differentiate between proteins interacting with DNA and proteins nonspecifically adsorbed to the surface.[9] Moreover, DNA curtains directly visualize individual protein–DNA interactions and track protein movement along DNA in real time. This reveals important behaviors such as binding kinetics, translocation, and target searching mechanisms.[19] Furthermore, DNA curtains can be modified into different configurations and work with a variety of DNA types, such as dsDNA and ssDNA.[19]

Limitations

DNA-curtain techniques are highly effective for conducting single-molecule studies. However, they have several limitations depending on the specific approach used. Traditional DNA curtains face several technical challenges, especially in creating effective lipid diffusion barriers. This process often requires specialized knowledge and expensive equipment.[19] Additionally, these systems are susceptible to stability issues and defects during the preparation of supported lipid bilayers. On the other hand, suspended DNA curtains face challenges due to their low throughput and the significant overlap of individual DNA molecules on nanowires.[19] This overlap makes imaging and analysis more complex. Additionally, Soft DNA curtains can be significantly affected by the non-specific binding of DNA-binding proteins to the protein-nanopatterned glass surface.[19] This unwanted interaction can complicate the measurement of protein-DNA interactions, leading to inaccurate results. Furthermore, the standard in vitro buffers used in DNA-curtain experiments do not accurately replicate the crowded biochemical environment present in living cells.[9]  

Recent Advances

Recent advances in DNA curtain-based single-molecule assays include the development of chromatin curtains, which enable direct visualization of nucleosome–protein interactions.[30] Also, this method has been extended beyond DNA substrates through the development of actin filament curtains, enabling visualization of cytoskeletal protein assembly at the single-molecule level.[31] Furthermore, UV lithography has been introduced as a more accessible and scalable method for fabricating lipid diffusion barriers.[5] This development makes it easier and more efficient to construct DNA curtains. Another advancement is the introduction of multichannel flow cells, which facilitate ATP titration experiments within a single experimental setup.[32]These flow cells simplify the process and enhance the ability to conduct multiple tests simultaneously. Another important advancement is the use of cell-derived extracts to mimic the crowded intracellular environment in vitro.[9] This strategy more accurately reflects physiological conditions. Therefore, this method has enabled more realistic single-molecule studies, such as DNA replication using Xenopus cell-free extracts[33] and spliceosome assembly using whole-cell extracts.[34]

See also

References

  1. ↑ 1.0 1.1 1.2 1.3 Ma, C.J.; Steinfeld, J.B.; Greene, E.C. (2017). "Single-Stranded DNA Curtains for Studying Homologous Recombination". Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods. Methods in Enzymology. 582. Elsevier. pp. 193–219. doi:10.1016/bs.mie.2016.08.005. ISBN 978-0-12-809310-8. PMC 7229809 Check |pmc= value (help). PMID 28062035. Search this book on
  2. ↑ 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 Greene, Eric C.; Wind, Shalom; Fazio, Teresa; Gorman, Jason; Visnapuu, Mari-Liis (2010). "DNA Curtains for High-Throughput Single-Molecule Optical Imaging". Single Molecule Tools: Fluorescence Based Approaches, Part A. Methods in Enzymology. 472. Elsevier. pp. 293–315. doi:10.1016/s0076-6879(10)72006-1. ISBN 978-0-12-374954-3. PMC 7233429 Check |pmc= value (help). PMID 20580969. Search this book on
  3. ↑ 3.0 3.1 3.2 Fazio, Teresa; Visnapuu, Mari-Liis; Wind, Shalom; Greene, Eric C. (2008-09-16). "DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging". Langmuir. 24 (18): 10524–10531. doi:10.1021/la801762h. ISSN 0743-7463. PMC 3033740. PMID 18683960.
  4. ↑ 4.0 4.1 4.2 4.3 Gibb, Bryan; Silverstein, Tim D.; Finkelstein, Ilya J.; Greene, Eric C. (2012-09-18). "Single-Stranded DNA Curtains for Real-Time Single-Molecule Visualization of Protein–Nucleic Acid Interactions". Analytical Chemistry. 84 (18): 7607–7612. Bibcode:2012AnaCh..84.7607G. doi:10.1021/ac302117z. ISSN 0003-2700. PMC 3475199. PMID 22950646.
  5. ↑ 5.0 5.1 Gallardo, Ignacio F.; Pasupathy, Praveenkumar; Brown, Maxwell; Manhart, Carol M.; Neikirk, Dean P.; Alani, Eric; Finkelstein, Ilya J. (2015-09-22). "High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging". Langmuir. 31 (37): 10310–10317. doi:10.1021/acs.langmuir.5b02416. ISSN 0743-7463. PMC 4624423. PMID 26325477.
  6. ↑ 6.0 6.1 Kopu̅stas, Aurimas; Ivanovaitė, Šaru̅nė; Rakickas, Tomas; Pocevičiu̅tė, Ernesta; Paksaitė, Justė; Karvelis, Tautvydas; Zaremba, Mindaugas; Manakova, Elena; Tutkus, Marijonas (2021-03-23). "Oriented Soft DNA Curtains for Single-Molecule Imaging". Langmuir. 37 (11): 3428–3437. doi:10.1021/acs.langmuir.1c00066. ISSN 0743-7463. PMC 8280724 Check |pmc= value (help). PMID 33689355 Check |pmid= value (help).
  7. ↑ 7.0 7.1 7.2 Cha, Jongjin; Lee, Ja Yil (2021-03-01). "A novel high-throughput single-molecule technique: DNA curtain". Journal of the Korean Physical Society. 78 (5): 442–448. Bibcode:2021JKPS...78..442C. doi:10.1007/s40042-020-00031-9. ISSN 1976-8524.
  8. ↑ 8.00 8.01 8.02 8.03 8.04 8.05 8.06 8.07 8.08 8.09 8.10 8.11 8.12 8.13 8.14 8.15 8.16 Collins, Bridget E.; Ye, Ling F.; Duzdevich, Daniel; Greene, Eric C. (2014). "DNA curtains". Quantitative Imaging in Cell Biology. Methods in Cell Biology. 123. Elsevier. pp. 217–234. doi:10.1016/b978-0-12-420138-5.00012-4. ISBN 978-0-12-420138-5. Retrieved 2025-11-27. Search this book on
  9. ↑ 9.00 9.01 9.02 9.03 9.04 9.05 9.06 9.07 9.08 9.09 9.10 9.11 Zhao, Yiling; Jiang, Yanzhou; Qi, Zhi (2017-04-20). "Visualizing biological reaction intermediates with DNA curtains". Journal of Physics D: Applied Physics. 50 (15): 153001. Bibcode:2017JPhD...50o3001Z. doi:10.1088/1361-6463/aa59cf. ISSN 0022-3727.
  10. ↑ Granéli, Annette; Yeykal, Caitlyn C.; Prasad, Tekkatte Krishnamurthy; Greene, Eric C. (2006-01-01). "Organized Arrays of Individual DNA Molecules Tethered to Supported Lipid Bilayers". Langmuir. 22 (1): 292–299. doi:10.1021/la051944a. ISSN 0743-7463. PMID 16378434.
  11. ↑ Visnapuu, Mari-Liis; Fazio, Teresa; Wind, Shalom; Greene, Eric C. (2008-10-07). "Parallel arrays of geometric nanowells for assembling curtains of DNA with controlled lateral dispersion". Langmuir. 24 (19): 11293–11299. doi:10.1021/la8017634. ISSN 0743-7463. PMC 2748852. PMID 18788761.
  12. ↑ 12.0 12.1 12.2 Wang, Feng; Redding, Sy; Finkelstein, Ilya J.; Gorman, Jason; Reichman, David R.; Greene, Eric C. (2013). "The promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion". Nature Structural & Molecular Biology. 20 (2): 174–181. doi:10.1038/nsmb.2472. ISSN 1545-9985. PMC 3565103. PMID 23262491.
  13. ↑ 13.0 13.1 Crickard, J. Brooks (2023). "Single Molecule Imaging of DNA–Protein Interactions Using DNA Curtains". In Simoes-Costa, Marcos. DNA-Protein Interactions. Methods in Molecular Biology. 2599. New York, NY: Springer US. pp. 127–139. doi:10.1007/978-1-0716-2847-8_10. ISBN 978-1-0716-2846-1. PMC 10082465 Check |pmc= value (help). PMID 36427147 Check |pmid= value (help). Search this book on
  14. ↑ 14.0 14.1 Finkelstein, Ilya J.; Greene, Eric C. (2011). "Supported lipid bilayers and DNA curtains for high-throughput single-molecule studies". DNA Recombination. Methods in Molecular Biology (Clifton, N.J.). 745. pp. 447–461. doi:10.1007/978-1-61779-129-1_26. ISBN 978-1-61779-128-4. ISSN 1940-6029. PMC 3319767. PMID 21660710. Search this book on
  15. ↑ 15.0 15.1 15.2 Soniat, Michael M.; Myler, Logan R.; Schaub, Jeffrey M.; Kim, Yoori; Gallardo, Ignacio F.; Finkelstein, Ilya J. (2017). "Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination". DNA Repair Enzymes: Structure, Biophysics, and Mechanism. Methods in Enzymology. 592. pp. 259–281. doi:10.1016/bs.mie.2017.03.011. ISBN 978-0-12-812515-1. ISSN 1557-7988. PMC 5564670. PMID 28668123. Search this book on
  16. ↑ Duzdevich, Daniel; Warner, Megan D.; Ticau, Simina; Ivica, Nikola A.; Bell, Stephen P.; Greene, Eric C. (2015-05-07). "The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level". Molecular Cell. 58 (3): 483–494. doi:10.1016/j.molcel.2015.03.017. ISSN 1097-2765. PMC 4427541. PMID 25921072.
  17. ↑ 17.0 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 Gorman, Jason; Wang, Feng; Redding, Sy; Plys, Aaron J.; Fazio, Teresa; Wind, Shalom; Alani, Eric E.; Greene, Eric C. (2012-11-06). "Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair". Proceedings of the National Academy of Sciences. 109 (45): E3074–E3083. doi:10.1073/pnas.1211364109. PMC 3494904. PMID 23012240.
  18. ↑ Crickard, J. Brooks; Xue, Chaoyou; Wang, Weibin; Kwon, Youngho; Sung, Patrick; Greene, Eric C. (2019-05-21). "The RecQ helicase Sgs1 drives ATP-dependent disruption of Rad51 filaments". Nucleic Acids Research. 47 (9): 4694–4706. doi:10.1093/nar/gkz186. ISSN 1362-4962. PMC 6511845 Check |pmc= value (help). PMID 30916344.
  19. ↑ 19.00 19.01 19.02 19.03 19.04 19.05 19.06 19.07 19.08 19.09 19.10 19.11 19.12 19.13 19.14 19.15 19.16 19.17 19.18 19.19 19.20 19.21 19.22 19.23 19.24 19.25 19.26 Kopūstas, Aurimas; Zaremba, Mindaugas; Tutkus, Marijonas (2022-01-11). "DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level". Applied Nano. 3 (1): 16–41. doi:10.3390/applnano3010002. ISSN 2673-3501.
  20. ↑ Visnapuu, Mari-Liis; Greene, Eric C. (2009). "Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition". Nature Structural & Molecular Biology. 16 (10): 1056–1062. doi:10.1038/nsmb.1655. ISSN 1545-9985. PMC 2779460. PMID 19734899.
  21. ↑ Axelrod, D (1981-04-01). "Cell-substrate contacts illuminated by total internal reflection fluorescence". The Journal of Cell Biology. 89 (1): 141–145. doi:10.1083/jcb.89.1.141. ISSN 0021-9525. PMC 2111781. PMID 7014571.
  22. ↑ Gorman, Jason; Greene, Eric C (2008). "Visualizing one-dimensional diffusion of proteins along DNA". Nature Structural & Molecular Biology. 15 (8): 768–774. doi:10.1038/nsmb.1441. ISSN 1545-9993. PMID 18679428.
  23. ↑ Ganji, Mahipal; Shaltiel, Indra A.; Bisht, Shveta; Kim, Eugene; Kalichava, Ana; Haering, Christian H.; Dekker, Cees (2018-04-06). "Real-time imaging of DNA loop extrusion by condensin". Science. 360 (6384): 102–105. Bibcode:2018Sci...360..102G. doi:10.1126/science.aar7831. ISSN 0036-8075. PMC 6329450. PMID 29472443.
  24. ↑ Kong, Muwen; Cutts, Erin E.; Pan, Dongqing; Beuron, Fabienne; Kaliyappan, Thangavelu; Xue, Chaoyou; Morris, Edward P.; Musacchio, Andrea; Vannini, Alessandro; Greene, Eric C. (2020). "Human Condensin I and II Drive Extensive ATP-Dependent Compaction of Nucleosome-Bound DNA". Molecular Cell. 79 (1): 99–114.e9. doi:10.1016/j.molcel.2020.04.026. PMC 7335352 Check |pmc= value (help). PMID 32445620 Check |pmid= value (help).
  25. ↑ von Hippel, P H; Berg, O G (1989). "Facilitated Target Location in Biological Systems". Journal of Biological Chemistry. 264 (2): 675–678. doi:10.1016/S0021-9258(19)84994-3. PMID 2642903.
  26. ↑ Gorman, Jason; Chowdhury, Arindam; Surtees, Jennifer A.; Shimada, Jun; Reichman, David R.; Alani, Eric; Greene, Eric C. (2007). "Dynamic Basis for One-Dimensional DNA Scanning by the Mismatch Repair Complex Msh2-Msh6". Molecular Cell. 28 (3): 359–370. doi:10.1016/j.molcel.2007.09.008. PMC 2953642. PMID 17996701.
  27. ↑ Finkelstein, Ilya J.; Visnapuu, Mari-Liis; Greene, Eric C. (2010). "Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase". Nature. 468 (7326): 983–987. Bibcode:2010Natur.468..983F. doi:10.1038/nature09561. ISSN 1476-4687. PMC 3230117. PMID 21107319.
  28. ↑ Lee, Ja Yil; Finkelstein, Ilya J.; Crozat, Estelle; Sherratt, David J.; Greene, Eric C. (2012-04-24). "Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK". Proceedings of the National Academy of Sciences. 109 (17): 6531–6536. Bibcode:2012PNAS..109.6531L. doi:10.1073/pnas.1201613109. PMC 3340036. PMID 22493241.
  29. ↑ Courtemanche, Naomi; Lee, Ja Yil; Pollard, Thomas D.; Greene, Eric C. (2013-06-11). "Tension modulates actin filament polymerization mediated by formin and profilin". Proceedings of the National Academy of Sciences. 110 (24): 9752–9757. Bibcode:2013PNAS..110.9752C. doi:10.1073/pnas.1308257110. ISSN 0027-8424. PMC 3683744. PMID 23716666.
  30. ↑ Visnapuu, Mari-Liis; Greene, Eric C. (2009). "Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition". Nature Structural & Molecular Biology. 16 (10): 1056–1062. doi:10.1038/nsmb.1655. ISSN 1545-9985. PMC 2779460. PMID 19734899.
  31. ↑ Courtemanche, Naomi; Lee, Ja Yil; Pollard, Thomas D.; Greene, Eric C. (2013-06-11). "Tension modulates actin filament polymerization mediated by formin and profilin". Proceedings of the National Academy of Sciences. 110 (24): 9752–9757. Bibcode:2013PNAS..110.9752C. doi:10.1073/pnas.1308257110. ISSN 0027-8424. PMC 3683744. PMID 23716666.
  32. ↑ Robison, Aaron D.; Finkelstein, Ilya J. (2014-05-06). "Rapid Prototyping of Multichannel Microfluidic Devices for Single-Molecule DNA Curtain Imaging". Analytical Chemistry. 86 (9): 4157–4163. Bibcode:2014AnaCh..86.4157R. doi:10.1021/ac500267v. ISSN 0003-2700. PMID 24734940.
  33. ↑ Yardimci, Hasan; Loveland, Anna B.; van Oijen, Antoine M.; Walter, Johannes C. (2012). "Single-molecule analysis of DNA replication in Xenopus egg extracts". Methods. 57 (2): 179–186. doi:10.1016/j.ymeth.2012.03.033. PMC 3427465. PMID 22503776.
  34. ↑ Hoskins, Aaron A.; Friedman, Larry J.; Gallagher, Sarah S.; Crawford, Daniel J.; Anderson, Eric G.; Wombacher, Richard; Ramirez, Nicholas; Cornish, Virginia W.; Gelles, Jeff; Moore, Melissa J. (2011-03-11). "Ordered and Dynamic Assembly of Single Spliceosomes". Science. 331 (6022): 1289–1295. Bibcode:2011Sci...331.1289H. doi:10.1126/science.1198830. ISSN 0036-8075. PMC 3086749. PMID 21393538.

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