Transchip: Single-molecule detection of transcriptional elongation complexes

Wu, Tian; Schwartz, David C.

doi:10.1016/j.ab.2006.10.042

Cited by 10 publications

(14 citation statements)

References 47 publications

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“…An EC may consist a long strand of nascent RNA produced from a single round of transcription [24], while the majority of ECs we observed consist of a "supercomplex" which resulted from multiple rounds of transcription from the same promoter being packed together. The molecular component and structure for "supercomplex" will be discussed in greater detail in later sections of this paper.…”

Section: Transcription Product Detection By Fluorescence Microscopymentioning

confidence: 95%

“…Our previous results showed that imaged punctates were ECs being paused or arrested at the DNA backbone during the transcription process under certain experimental conditions [24]. An EC may consist a long strand of nascent RNA produced from a single round of transcription [24], while the majority of ECs we observed consist of a "supercomplex" which resulted from multiple rounds of transcription from the same promoter being packed together.…”

Section: Transcription Product Detection By Fluorescence Microscopymentioning

confidence: 96%

“…Image collection was performed by a fully automated image acquisition system developed by our laboratory (Channelcollect) [11]. Image analysis was done similar to the process described previously [24]. Transcription products were manually marked and analyzed with the image editing software, Omari, which assigns contour length in pixels for each DNA fragment and EC, as well as integrated fluorescence intensity for each fragment and EC.…”

Section: Staining Fluorescence Image Acquisition and Processingmentioning

confidence: 99%

“…Recent applications of the OM system [19;20;21;22] enabled comparative analysis for entire genomes [23], and supported imaging of in vitro transcription products associated with templates mounted on OM surfaces for visualization, quantitation, mapping locations and statistical analysis [24].…”

Section: Introductory Statementmentioning

confidence: 99%

“…Such errors may also explain possible outliers appearing as transcription products upstream from the promoter, in Figure 3a, left to the 15 kb bar, where ECs are not expected. Additionally, non-specific transcription could also contribute to the presence of punctates at unexpected positions [24]. …”

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confidence: 99%

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Imaging and analysis of transcription on large, surface-mounted single template DNA molecules

Schwartz

2008

Analytical Biochemistry

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A surface-based approach is presented for the transcriptional analyses of large, single DNA molecule templates and their imaged reaction products by RNA polymerase (RNAP). Results showed surfaces with a charge density supporting stretching of single DNA molecules to 70-80% of their full contour length were ideal for analysis of T7 RNAP transcription complexes on bound single template DNAs. Such DNA molecules were shown to sustain efficient transcription reactions and analysis, which enabled localization of transcription complexes on templates at kilobase resolution. Direct labeling of nascent RNA transcripts by the incorporation of a second fluorochrome into DNA templates promotes more robust and sensitive detection of punctuates. Further characterization by RNase digestions, AFM studies, and fluoro-immuno-labeling revealed a "supercomplex" structure within a punctate where elongation complexes (ECs) aggregate through entanglement of DNA and RNA strands from individual ternary ECs. We have proposed mechanisms that underlie the supercomplex formation process. Whereas supercomplexes develop naturally in free solution, spatial constraints involved in a topologically limited system where template DNA was bound to the surface may facilitate the assembling process by stalling transcriptional elongation. Keywordspromoter detection; functionalized surfaces; fluorescence microscopy; T7 RNA polymerase; AFM Introductory StatementSingle molecule studies of transcription have revealed remarkable insights into individual steps, interactions, and associated reactions, and have been recently reviewed in [1;2]. For direct visualization and tracking of RNAP action, a key step involves effective immobilization and manipulation of molecules facing chain dynamics and Brownian fluctuations. Several early approaches have served such purpose by the immobilization of RNAP to surface substrates and monitoring beads that were attached to individual DNA molecules were being transcribed [3], and by use of patterned DNA templates on surfaces enabling tracking of fluorescently labeled polymerase perturbed by fluid flow [4]. Similarly, loose attachment of DNA templates on mica surfaces allowed time lapse AFM imaging of RNAP action [5]. More recently, manipulation of single DNA molecules by optical or magnetic tweezers have provided precise control and resolution for observing transcription [6;7;8] including basepair resolution during "stepping" [9].+ To whom correspondence should be addressed: dcschwartz@wisc.edu, . NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThis paper reports the basis for a high-throughput transcription analysis system that utilizes fluorescence imaging and analysis of transcription performed on template DNA molecules stretched and immobilized on surface substrates. Accordingly, to achieve high-throughput, immobilized individual DNA molecules must remain biochemically competent despite their presentation in a stretched linear form. This is important, since precise loc...

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Section: Transcription Product Detection By Fluorescence Microscopymentioning

confidence: 95%

Section: Transcription Product Detection By Fluorescence Microscopymentioning

confidence: 96%

Section: Staining Fluorescence Image Acquisition and Processingmentioning

confidence: 99%

Section: Introductory Statementmentioning

confidence: 99%

mentioning

confidence: 99%

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Imaging and analysis of transcription on large, surface-mounted single template DNA molecules

Schwartz

2008

Analytical Biochemistry

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Visualization of large elongated DNA molecules

et al. 2015

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Long and linear DNA molecules are the mainstream single-molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA-protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.

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Combining atomic force and fluorescence microscopy for analysis of quantum‐dot labeled protein–DNA complexes

Ebenstein

Gassman

Kim

et al. 2009

J of Molecular Recognition

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Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA.

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Transchip: Single-molecule detection of transcriptional elongation complexes

Cited by 10 publications

References 47 publications

Imaging and analysis of transcription on large, surface-mounted single template DNA molecules

Imaging and analysis of transcription on large, surface-mounted single template DNA molecules

Visualization of large elongated DNA molecules

Combining atomic force and fluorescence microscopy for analysis of quantum‐dot labeled protein–DNA complexes

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