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

Abstract

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...

Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point

“…46 Using the average FWHM to estimate resolution, 46 the extensional-flow stretching method may be able to discriminate between target sequences separated by as little as 2 kb, an improvement over the resolution reported for direct linear analysis (DLA), where DNA are entrained and stretched in a contraction flow. 16,47 For comparison, peak resolutions of 1-5 kb (based on FWHM) have recently been demonstrated [48][49][50] for ensembles of 100-600 slide-immobilized samples. Ultimately, any method based on optical detection for a population of molecules will likely be diffraction limited with a minimum resolution of $0.5 kb for fully extended stained DNA.…”

Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping

“…This is comparable to or better than previous single molecule target sequence position determination; for example resolutions of 1–5 kb, based on the full width at half maximum of populations of 100–600 slide-immobilized DNA molecules, have been recently reported by other single molecule techniques. 7–9 We previously demonstrated the use of cross slot flows for sequence detection using a biotinylated mutant of EcoRI attached to a NeutrAvidin coated fluorescent nanosphere, 21 however in general the creation of such fluorescent probes is expensive, time-consuming, and the biotinylation or labeling reactions may have low efficiency or interfere with the enzymatic activity. We note that in our earlier study, the error was higher and the precision slightly lower than that reported here; we believe the improved precision in target sequence location in the present case is due to the absence of the nanosphere, which leads to lower fluctuations in the length of the DE complex in this stagnation point flow (see Supplementary information Fig.…”

“…Restriction enzymes are routinely used for DNA modification and manipulation due to their ability to cut or cleave DNA at specific nucleotide sequences known as restriction or recognition sites. They are widely used in molecular biology, recombinant DNA technology, 1 gene mapping techniques [2][3][4][5][6][7][8][9][10] and DNA sequencing. 11,12 In optical restriction mapping, [3][4][5][6][7] for example, genomic length dsDNA is stretched and immobilized on a substrate, digested with a restriction enzyme, stained with a fluorescent dye, and the positions at which the enzyme has cut the DNA are used to identify the locations of the restriction sites.…”

DNA combing on low-pressure oxygen plasma modified polysilsesquioxane substrates for single-molecule studies

“…10,15 Schwartz and his coworkers used trimethylsilane and dimethyldiethoxysilane (DMDES) coated coverslides to render a hydrophobic surface for effective DNA combing. 16,17 Larson's group reported a novel protein assisted DNA immobilization (PADI) on micro-or nanofluidic surfaces, in which restriction enzymes or RNAP was used to anchor DNA molecules with limited surface interactions. 9 Other notable methods involve the use of biotin-streptavidin coupling, neutravidin coated surfaces, polystyrene coated coverslides, aminopropyltriethoxysilane (APTES) coated surface, receding meniscus on micro-structured polydimethylsiloxane (PDMS) substrates, etc., for DNA stretching and immobilization.…”