Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy.

Visualization of DNA-protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3'-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

Single-molecule studies of DNA transcription using atomic force microscopy.

Atomic force microscopy (AFM) can detect single biomacromolecules with a high signal-to-noise ratio on atomically flat biocompatible support surfaces, such as mica. Contrast arises from the innate forces and therefore AFM does not require imaging contrast agents, leading to sample preparation that is relatively straightforward. The ability of AFM to operate in hydrated environments, including humid air and aqueous buffers, allows structure and function of biological and biomolecular systems to be retained. These traits of the AFM are ensuring that it is being increasingly used to study deoxyribonucleic acid (DNA) structure and DNA-protein interactions down to the secondary structure level. This report focuses in particular on reviewing the applications of AFM to the study of DNA transcription in reductionist single-molecule bottom-up approaches. The technique has allowed new insights into the interactions between ribonucleic acid (RNA) polymerase to be gained and enabled quantification of some aspects of the transcription process, such as promoter location, DNA wrapping and elongation. More recently, the trend is towards studying the interactions of more than one enzyme operating on a single DNA template. These methods begin to reveal the mechanics of gene expression at the single-molecule level and will enable us to gain greater understanding of how the genome is transcribed and translated into the proteome.

Mechanically unfolding protein L using a laser-feedback-controlled cantilever.

Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is necessary both to explore the entire force spectrum and to characterize each species populated during unfolding. In the conventional AFM apparatus, force is applied to the protein construct through a compliant cantilever. This limits the dynamic range of the force spectrum that can be probed, and the cantilever recoil after unfolding may mask the presence of metastable intermediates. Here, we describe to our knowledge a new technique-constant-deflection AFM-in which the compliance of the AFM cantilever is removed. Using this technique, we show that protein L exhibits a more complex unfolding energy landscape than previously detected using the conventional technique. This technique is also able to detect the presence of a refolding intermediate whose formation is otherwise prevented by cantilever recoil.

CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.

DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting "CyDNA" displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties.

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned lambda(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

Studying silane mobility on hydrated mica using ambient AFM.

The mobility of the mono-functional aminosilane, aminopropyldimethylmethoxysilane (APDMMS), on mica has been investigated under ambient relative humidity (RH) using tapping-mode (TM) AFM. Silane layers were formed from vapour phase at various, controlled humidities and then imaged at ambient laboratory conditions (typically 30-40% RH). At low RH of formation (<25%) films without any resolvable sub-structure were formed, and these were stable to the imaging probe. At high RH of formation (>25%), where islands of phase II water are known to exist on mica, a two-phase domain structure was seen as two height levels and the surface area of the higher domains correlated with the RH. Sequential images taken over a 30-60 min time period show that these domains are mobile and at intermediate to high RH the domains coalesce under the influence of the scanning AFM tip. The results suggest the APDMMS resides at the air-water interface (rather than interacting with the mica) and that it preferentially interacts with the mobile phase II water as opposed to the phase I water tightly bound to the mica.

Unravelling the design principles for single protein mechanical strength.

In recent years single molecule manipulation techniques have improved to the extent that measurements of the mechanical strength of single proteins can now be undertaken routinely. This powerful new tool, coupled with theoretical frameworks to characterise the unfolding process, has enabled significant progress to be made in understanding the physical mechanisms that underlie protein mechanical strength. These design concepts have allowed the search for proteins with novel, mechanically strong folds to be automated and for previously mechanically characterised proteins to be engineered rationally. Methods to achieve the latter are diverse and include re-engineering of specific hydrophobic core residues, changing solvent conditions and the 'cross-linking' of side-chains that are separated in the rate-limiting unfolding transition. Predicting the mechanical behaviour of larger proteins and those with more complex structures remains a significant challenge while on-going instrument development is beginning to allow the examination of mechanical strength of protein across a wide range of force loading rates. The integral role of force in biology and the potential for exploitation of catalytic and structural proteins as functional bio-materials makes this a particularly important area of research.

DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes.

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping.

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1-3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 +/- 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.

Imaging RNA polymerase-amelogenin gene complexes with single molecule resolution using atomic force microscopy.

The AMELX gene encoding the enamel matrix protein, amelogenin, is located within (and in the opposite orientation to) the first intron of the ARHGAP6 gene, which encodes a GTPase-activating protein. The orientation of these two genes with respect to each other raises the possibility that they may undergo simultaneous convergent transcription during amelogenesis. The aim of this study was to use atomic force microscopy (AFM) to study a transcriptionally active amelogenin DNA template and to investigate the binding of RNA polymerase to convergently aligned promoters. Images of RNA polymerases stalled on DNA templates were obtained following incubation of the template with RNA polymerases and ribonucleotide triphosphates. A linear DNA template incorporating an intact rat amelogenin cDNA flanked by convergently aligned coliphage T7 and T3 promoters was constructed and shown to be transcriptionally active in vitro. Atomic force microscopy images of transcription complexes revealed globular structures, corresponding to single RNA polymerase molecules bound at specific locations on the DNA templates. These results indicate that AFM allows the visualization of individual RNA polymerases on DNA templates, offering a realistic approach to investigating the concept of convergent transcription of nested genes, which may lead to an understanding of whether the simultaneous expression of AMELX and ARHGAP6 is possible during the formation of tooth enamel.

Formation of aminosilane-functionalized mica for atomic force microscopy imaging of DNA.

Factors affecting the functionalization of mica with aminosilanes, in particular, aminopropyltriethoxysilane (APTES-mica), formed from the vapor phase have been systematically studied. The relative humidity (RH) during vapor deposition has been varied, and postdeposition treatment through baking has been used, as well as the comparison of mono and trifunctionality, to investigate how optimal surfaces for AFM imaging of DNA are formed. It is found that the stability of the APTES layers is a consequence of lateral polymerization and not covalent attachment to the mica substrate. At low RH (<25%), DNA adopts an open, well-resolved conformation, whereas at >25% RH, DNA surface-induced condensation occurs. Contact mode AFM scratching experiments show that two main structures of the silane layer exist at different humidity: a monolayer exists at RH < 25%, and a bilayer structure exists at RH > 25%. Finally, structural changes that these two layer types undergo after baking at 150 degrees C were investigated by AFM and X-ray photoelectron spectroscopy (XPS), and these now prevented DNA from binding to the APTES-mica, except in the presence of Mg(II) ions.