Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca.

Fiber Bragg grating inscription assisted by a spatial light modulator.

In this Letter, we proposed a new technique for point-by-point fiber Bragg grating (FBG) writing in a static fiber by using a spatial light modulator to control the position of the focal point inside the fiber core. Various types of short-length FBGs (uniform, phase-shifted, and apodized) were demonstrated by this inscription technique. Moreover, the capability to tailor the transverse dimension of a grating pitch from a single point to more complex shapes, such as a wide plane covering a whole fiber core or a transverse ring, was shown.

A Single-Molecule Surface-Based Platform to Detect the Assembly and Function of the Human RNA Polymerase II Transcription Machinery.

Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the biomolecule(s) of interest to a biocompatible surface. Here, we describe a robust surface passivation method by dense polymer brush grafting, based on optimized polyethylene glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system.

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently.

A general method to improve fluorophores for live-cell and single-molecule microscopy.

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores.

[ZINK IS ACTIVATOR OF ENTERAL CALCIUM METABOLISM].

Experiments on goats and rabbits showed that zinc supplement to the diet leads to calcium concentration rise in muscle, bone and blood of animals. However, this rise was not adequate to increase in.zinc consumption.

Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4.

Single-molecule tracking of the transcription cycle by sub-second RNA detection.

Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (∼10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts.

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution.

Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy.

Magnetic trap construction.

In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead.

Single-molecule studies using magnetic traps.

In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead.

Static and kinetic site-specific protein-DNA photocrosslinking: analysis of bacterial transcription initiation complexes.

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking - involving rapid-quench-flow mixing and pulsed-laser irradiation - permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes.

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.

Site-specific protein-DNA photo-cross-linking: analysis of structural organization of protein-DNA and multiprotein-DNA complexes.

Site-specific protein-DNA photo-cross-linking is able to define positions of proteins relative to DNA within large multiprotein-DNA complexes. Chemical and enzymatic reactions are used to prepare a DNA fragment containing a phenyl-azide photoactivatible cross-linking agent and an adjacent radiolabel incorporated at a single, defined DNA phosphate. The multiprotein-DNA complex of interest is formed using the site-specifically derivatized DNA fragment.

Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation.

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with approximately 1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximately 5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides.

Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.

We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding sigma(70)-(RNA polymerase core), sigma(70)-DNA, and sigma(70)-RNA interactions. The positions of sigma(70) regions 1.

Structural organization of the RNA polymerase-promoter open complex.

We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.