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. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.

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. The increased biocompatibility achieved, compared with PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macromolecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.

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. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

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. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy.

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼ 4-µm-deep volume, with lateral and axial localization precisions of ∼ 20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.

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. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

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. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001.

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. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

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. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 pN. In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. In this article, we describe the principle of the magnetic trap, as well as its use in the measurement of the elastic properties of DNA and the study of DNA-protein interactions.

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. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 picoNewtons (pN). In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. This protocol describes a procedure for building and using a magnetic trap. It gives a method for constructing a microchamber suitable for magnetic tweezers studies, including antibody coating and passivation. It also describes a series of simple steps to achieve end-labeling of DNA anchoring fragments. One anchoring fragment is biotin-labeled and the other is labeled with digoxigenin. The anchoring fragments are then digested and ligated to a central DNA region containing the sequence of interest. The biotinylated DNA is adsorbed onto streptavidin-coated magnetic beads, and the DNA-bead mixture attaches specifically to the antidigoxigenin-coated surface of the microchamber.

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. The complex is then UV-irradiated, initiating covalent cross-linking with proteins in direct physical proximity to the cross-linking agent. Extensive nuclease digestion is performed to eliminate uncross-linked DNA and convert cross-linked DNA to a cross-linked, radiolabeled nucleotide "tag." The "tagged" proteins are identified, usually by denaturing polyacrylamide gel electrophoresis followed by autoradiography. The procedure is performed in a systematic fashion: At least 10 derivatized DNA fragments, each having the cross-linking agent incorporated at a defined DNA phosphate within the region of interest, are analyzed. The results of the procedure define the translational positions of proteins relative to the DNA sequence. Plotted on a three-dimensional representation of a DNA helix, the results also define the rotational orientations of proteins relative to the DNA helix axis and the groove orientations of proteins relative to the DNA major and minor grooves. Here, we present a detailed protocol for the cross-linking of protein-DNA complexes immobilized on streptavidin-coated paramagnetic beads ("on-bead" cross-linking).

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. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.