Double-stranded DNA Templates Research Articles

Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse

Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. Alternative ways to provide the replacement DNA sequence, method of Cas9 delivery, and tethering the template sequence to Cas9 or the guide RNA (gRNA) have all been tested in an effort to maximize homology-directed repair for precise modification of the genome. We present two CRISPR/Cas9 methods that have been used to successfully generate large and complex gene-edits in mouse. In the first method, the Cas9 enzyme is used in conjunction with two sgRNAs and a long single-stranded DNA (lssDNA) template prepared by an alternative protocol. The second method utilizes a tethering approach to couple a biotinylated, double-stranded DNA (dsDNA) template to a Cas9-streptavidin fusion protein.•Alternative method for generating long, single-stranded DNA templates for CRISPR/Cas9 editing.•Demonstration that using two sgRNAs with Cas9-streptavidin/biotinylated-dsDNA is feasible for large DNA modifications.

Isothermal amplification using modified primers for rapid electrochemical analysis of coeliac disease associated DQB1*02 HLA allele

DNA biosensors are attractive tools for genetic analysis as there is an increasing need for rapid and low-cost DNA analysis, primarily driven by applications in personalized pharmacogenomics, clinical diagnostics, rapid pathogen detection, food traceability and forensics. A rapid electrochemical genosensor detection methodology exploiting a combination of modified primers for solution-phase isothermal amplification, followed by rapid detection via hybridization on gold electrodes is reported. Modified reverse primers, exploiting a C18 spacer between the primer-binding site and an engineered single stranded tail, are used in a recombinase polymerase amplification reaction to produce an amplicon with a central duplex flanked by two single stranded tails. These tails are designed to be complementary to a gold electrode tethered capture oligo probe as well as a horseradish peroxidase labelled reporter oligo probe. The time required for hybridization of the isothermally generated amplicons with each of the immobilized and reporter probes was optimised to be 2 min, in both cases. The effect of amplification time and the limit of detection were evaluated using these hybridization times for both single stranded and double stranded DNA templates. The best detection limit of 70 fM for a ssDNA template was achieved using 45 min amplification, whilst for a dsDNA template, just 30 min amplification resulted in a slightly lower detection limit of 14 fM, whilst both 20 and 45 min amplification times were observed to provide detection limits of 71 and 72 fM, respectively, but 30 and 45 min amplification resulted in a much higher signal and sensitivity. The genosensor was applied to genomic DNA and real patient and control blood samples for detection of the coeliac disease associated DQB1*02 HLA allele, as a model system, demonstrating the possibility to carry out molecular diagnostics, combining amplification and detection in a rapid and facile manner.

In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI

In this protocol, two oligonucleotides are used to prime DNA synthesis by a high-fidelity polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and have the same starting and ending positions on opposite strands of the plasmid DNA. The entire lengths of both strands of the plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutated plasmid containing staggered nicks on opposite strands. Because of the amount of template DNA used in the amplification reaction, the background of transformed colonies containing wild-type plasmid DNA can be quite high unless steps are taken to enrich for mutant molecules. In this protocol, the products of the linear amplification reaction are treated with the restriction enzyme DpnI, which specifically cleaves fully methylated GMe6ATC sequences. DpnI will therefore digest the bacterially generated DNA used as template for amplification, but it will not digest DNA synthesized during the course of the reaction in vitro. DpnI-resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli cells to antibiotic resistance. Because the method works well with virtually any plasmid of moderate size (<7 kb), it can be used to introduce mutations directly into full-length cDNAs and eliminates the need for subcloning into specialized vectors.

Infrared laser ablation sample transfer of tissue DNA for genomic analysis.

Infrared (IR) laser ablation was used to remove material from tissue sections mounted on microscope slides, with subsequent capture in a solvent-containing microcentrifuge tube. Experiments conducted with a 3200-bp double-stranded plasmid DNA template demonstrated IR-laser ablation transfer of intact DNA. The transfer efficiency and the molecular integrity of the captured DNA were evaluated using Sanger sequencing, gel electrophoresis, and fluorimetric analysis. The plasmid DNA was reproducibly transferred with an efficiency of 59±3% at laser fluences of between 10 and 20kJ/m2 at a wavelength of 3μm. IR laser ablation sample transfer was then used to ablate and capture DNA from 50-μm-thick rat brain and kidney tissue sections. DNA was extracted from the captured material using five commercial DNA extraction kits that employed significantly divergent methodologies, with all kits recovering sufficient DNA for successful amplification by polymerase chain reaction (PCR). Four sets of primers were employed, targeting one region of the CYP 11b2 gene (376bp) and three different regions of the Snn1g gene (298, 168, and 281bp). The PCR results were not consistently reliable when using unpurified ablation samples; however, after extraction, all samples produced PCR products of the expected size. This work expands the sampling capabilities of IR laser ablation, demonstrating that DNA can be isolated from tissue samples for genomic assays. Due to the small size of the ablation regions (1mm2), this technique will be useful for sampling discrete cell populations from tissue sections. Graphical abstract Infrared laser ablation transfer of intact DNA from a tissue section.

Abstract PR02: Small molecules that specifically inhibit the D-loop activity of RAD51

Abstract RAD51 plays a central role in homologous recombination (HR), which maintains genome integrity. RAD51 is commonly overexpressed in cancer cells relative to normal tissue and is considered a therapeutic target in oncology. One potential challenge for targeting RAD51 pharmacologically is that it mediates functions in both double-strand DNA break (DSB) repair and stabilization of stalled replication forks. In order to distinguish compound-mediated effects on these RAD51 functions, we developed a novel class of RAD51 inhibitors. In contrast to many previously reported RAD51 inhibitors, we sought to develop compounds that do not inhibit RAD51's ability to bind single-stranded DNA (ssDNA). Instead, these compounds prevent RAD51-ssDNA nucleoprotein filaments from invading into homologous double-stranded DNA templates and forming D-loops. Our initial lead compound RI(dl)-1 (an abbreviation for RAD51 inhibitor of D-loop formation #1) is capable of blocking RAD51-mediated D-loop formation in biochemical assays, using concentrations that do not prevent RAD51 binding to ssDNA. An analog of this compound, termed RI(dl)-2, provides even better inhibition of RAD51's D-loop activity in biochemical systems (IC50 15.8 µM). RI(dl)-2 reduces HR activity in cells in dose ranges that do not stimulate single strand annealing (SSA) activity, which distinguishes it more generalized RAD51 inhibitors. RI(dl)-2 also sensitizes several cancer cell lines to radiation-induced death. We now present the results of more extensive structure activity relationship (SAR) optimizations aimed at further improving the potency and selectivity of this class of compounds. Several of these compounds are capable of delaying the timely resolution of radiation-induced RAD51 foci, even though initial RAD51 foci are apparently normal at earlier time points. Similar focus kinetics are observed for gamma-H2AX in identically treated cells, supporting the interpretation that these compounds permit RAD51 assembly at DSBs but prevent the completion of DSB repair. Unlike RI(dl)-2, some these newer compounds sensitize cancer cells to mitomycin C. These specialized RAD51 inhibitory compounds are attractive candidates for potential use as radiation or chemotherapy sensitizers, since they may exert fewer toxic risks compared to more generalized inhibitors of the HR repair machinery. They may also serve to help distinguish the functions of RAD51 DSB repair and replication stress tolerance, which may enable better understanding of RAD51's roles in maintaining genome stability. This abstract is also being presented as Poster A02. Citation Format: Brian Budke, Wei Lv, Werner Tueckmantel, Alan Kozikowski, Philip Connell. Small molecules that specifically inhibit the D-loop activity of RAD51 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR02.

Self-assembly of genetically encoded DNA-protein hybrid nanoscale shapes.

We describe an approach to bottom-up fabrication that allows integration of the functional diversity of proteins into designed three-dimensional structural frameworks. A set of custom staple proteins based on transcription activator-like effector proteins folds a double-stranded DNA template into a user-defined shape. Each staple protein is designed to recognize and closely link two distinct double-helical DNA sequences at separate positions on the template. We present design rules for constructing megadalton-scale DNA-protein hybrid shapes; introduce various structural motifs, such as custom curvature, corners, and vertices; and describe principles for creating multilayer DNA-protein objects with enhanced rigidity. We demonstrate self-assembly of our hybrid nanostructures in one-pot mixtures that include the genetic information for the designed proteins, the template DNA, RNA polymerase, ribosomes, and cofactors for transcription and translation.

Protein-folded DNA nanostructures

DNA Nanotechnology A wide variety of DNA nanostructures have been assembled by folding long DNA single strands with short DNA staples. However, such structures typically need annealing at elevated temperatures in order to form. To accommodate the formation of such structures in living cells, Praetorius and Dietz developed an approach in which custom protein staples based on transcription activator–like effector proteins fold double-stranded DNA templates (see the Perspective by Douglas). The structures folded into user-defined geometric shapes on the scale of tens to hundreds of nanometers. These nanostructures could self-assemble at room temperature in physiological buffers. Science , this issue p. [eaam5488][1]; see also p. [1261][2] [1]: /lookup/doi/10.1126/science.aam5488 [2]: /lookup/doi/10.1126/science.aam9541

Genetically Encoded DNA-Protein Hybrid Origami

Here we describe an approach to bottom-up fabrication with nanometer-precision that allows integrating the functional diversity of proteins in designed three-dimensional structural frameworks. We reimagined the successful DNA origami design principle using a set of custom staple proteins to fold a double-stranded DNA template into a user-defined shape. Each staple protein recognizes two distinct double-helical DNA sequences and can carry additional functionalities. The staple proteins we present here are based on the transcription activator-like (TAL) effector proteins. Due to their repetitive structure these proteins offer a unique programmability that enables us to construct numerous staple proteins targeting any desired DNA sequence. Our approach is general, meaning that many different objects may be created using the same set of rules, and it is modular, because components can be modified or exchanged individually. We present rules for constructing megadalton-scale DNA-protein hybrid nanostructures; introduce important structural motifs, such as curvature, corners, and vertices; describe principles for creating multi-layer DNA-protein objects with enhanced rigidity; and demonstrate the possibility to combine our DNA-protein hybrid origami with conventional DNA nanotechnology. Since all components can be encoded genetically, our structures should be amenable to biotechnological mass-production. Moreover, since the target objects can self-assemble at room temperature in near-physiological buffer, our hybrid origami may also provide an attractive method to realize positioning and scaffolding tasks in vivo. We expect our method to find application both in scaffolding protein functionalities and in manipulating the spatial arrangement of genomic DNA.

One facile fluorescence strategy for sensitive detection of endonuclease activity using DNA-templated copper nanoclusters as signal indicators

Assay of endonuclease activity plays an important role in biologic and medicinal study. Herein, a simple and facile strategy was developed for detecting endonuclease activity. In this strategy, poly(AT-TA) double-stranded DNA containing restriction cutting site for EcoRI endonuclease was rationally designed and acted as the substrate of EcoRI and the template for copper nanoclusters (CuNCs). In the presence of EcoRI, the double-stranded DNA was cleaved, and then the introduction of Cu2+ and ascorbate cannot form the fluorescent CuNCs due to the lack of the double-stranded DNA templates. The endonuclease actvity was quantified by monitoring the change in fluorescence intensity. Under the optimized conditions, the fluorescence change was linear with EcoRI concentration in the range from 0.002U/μL to 0.1U/μL with the detection limit of 0.00087U/μL. This method was easy and convenient to operate in homogeneous solution, and the whole assay process can be completed in a single tube.

Metal‐Ion‐Triggered Exonuclease III Activity for the Construction of DNA Colorimetric Logic Gates

The logic system is obtained by using a series of double-stranded (ds) DNA templates with mismatched base pairs (T-T or C-C) and ion-modulated exonuclease III (Exo III) activity, in which the Exo III cofactors, Hg(2+) and Ag(+) ions, are used as inputs for the activation of the respective scission of Exo III based on the formation of T-Hg(2+) -T or C-Ag(+) -C base pairs. Additionally, two kinds of signal probes are utilized to transduce the logic operations. One is the two split G-rich DNA strands that are used to design the OR, AND, INHIBIT, and XOR gates, whereas the other is the self-assembled split G-quadruplex structure to construct NOR, NAND, IMPLICATION, and XNOR operations based on DNA hybridization and strand displacement. In the presence of hemin, the split G-quadruplex biocatalyzes the formation of a colored product, which is an output signal for the different logic gates. Thus, we have constructed a complete set of colorimetric DNA logic gates based on the Exo III and split G-quadruplex for the first time. In addition, we are able to effortlessly recognize the logic output signals by the naked eye and their simplicity and cost-effective design is the most apparent feature for the logic gates developed in this work.

Nanoparticles Affect PCR Primarily via Surface Interactions with PCR Components: Using Amino-Modified Silica-Coated Magnetic Nanoparticles as a Main Model.

Nanomaterials have been widely reported to affect the polymerase chain reaction (PCR). However, many studies in which these effects were observed were not comprehensive, and many of the proposed mechanisms have been primarily speculative. In this work, we used amino-modified silica-coated magnetic nanoparticles (ASMNPs, which can be collected very easily using an external magnetic field) as a model and compared them with gold nanoparticles (AuNPs, which have been studied extensively) to reveal the mechanisms by which nanoparticles affect PCR. We found that nanoparticles affect PCR primarily by binding to PCR components: (1) inhibition, (2) specifity, and (3) efficiency and yield of PCR are impacted. (1) Excess nanomaterials inhibit PCR by adsorbing to DNA polymerase, Mg(2+), oligonucleotide primers, or DNA templates. Nanoparticle surface-active groups are particularly important to this effect. (2, a) Nanomaterials do not inhibit nonspecific amplification products caused by false priming as previously surmised. It was shown that relatively low concentrations of nanoparticles inhibited the amplification of long amplicons, and increasing the amount of nanoparticles inhibited the amplification of short amplicons. This concentration phenomenon appears to be the result of the formation of "joints" upon the adsorption of ASMNPs to DNA templates. (b) Nanomaterials are able to inhibit nonspecific amplification products due to incomplete amplification by preferably adsorbing single-stranded incomplete amplification products. (3) Some types of nanomaterials, such as AuNPs, enhance the efficiency and yield of PCR because these types of nanoparticles can adsorb to single-stranded DNA more strongly than to double-stranded DNA. This behavior assists in the rapid and thorough denaturation of double-stranded DNA templates. Therefore, the interaction between the surface of nanoparticles and PCR components is sufficient to explain most of the effects of nanoparticles on PCR.

Surface Enzyme Chemistries for Ultrasensitive Microarray Biosensing with SPR Imaging.

The sensitivity and selectivity of surface plasmon resonance imaging (SPRI) biosensing with nucleic acid microarrays can be greatly enhanced by exploiting various nucleic acid ligases, nucleases, and polymerases that manipulate the surface-bound DNA and RNA. We describe here various examples from each of these different classes of surface enzyme chemistries that have been incorporated into novel detection strategies that either drastically enhance the sensitivity of or create uniquely selective methods for the SPRI biosensing of proteins and nucleic acids. A dual-element generator–detector microarray approach that couples a bioaffinity adsorption event on one microarray element to nanoparticle-enhanced SPRI measurements of nucleic acid hybridization adsorption on a different microarray element is used to quantitatively detect DNA, RNA, and proteins at femtomolar concentrations. Additionally, this dual-element format can be combined with the transcription and translation of RNA from surface-bound double-stranded DNA (dsDNA) templates for the on-chip multiplexed biosynthesis of aptamer and protein microarrays in a microfluidic format; these microarrays can be immediately used for real-time SPRI bioaffinity sensing measurements.

TRF1 and TRF2 differentially modulate Rad51-mediated telomeric and nontelomeric displacement loop formation in vitro.

A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.

An In Vitro Selection Protocol for Threose Nucleic Acid (TNA) Using DNA Display.

Threose nucleic acid (TNA) is an unnatural genetic polymer composed of repeating threofuranosyl sugars linked by 2' and 3' phosphodiester bonds. TNA is capable of forming antiparallel Watson-Crick duplex structures in a self-pairing mode, and can also cross-pair opposite complementary strands of DNA and RNA. The solution NMR structure of a self-complementary TNA duplex reveals that TNA adopts an A-form helical structure, which explains its ability to exchange genetic information with natural genetic polymers. In a recent advance, a TNA aptamer was evolved from a pool of random sequences using an engineered polymerase that can copy DNA sequences into TNA. This unit details the steps required to evolve functional TNA molecules in the laboratory using a method called DNA display. Using this approach, TNA molecules are physically linked to their encoding double-stranded DNA template. By linking TNA phenotype with DNA genotype, one can select for TNA molecules with a desired function and recover their encoding genetic information by PCR amplification. Each round of selection requires ∼3 days to complete and multiple rounds of selection and amplification are required to generate functional TNA molecules.

Mechanism of gold nanoparticle induced simultaneously increased PCR efficiency and specificity

Gold nanoparticles (AuNPs) have been reported to induce faster heat transfer of polymerase chain reaction (PCR) solution to enhance PCR efficiency. AuNPs can also increase the specificity of PCR by functioning analogously to single-stranded DNA binding protein (SSB). However, the simple structure of AuNPs makes it difficult to determine how AuNPs affect PCR efficiency and specificity. This study aimed to elucidate the effect of AuNPs on PCR efficiency by altering the denaturation times and annealing temperatures. In addition, comparative study of the effect of AuNPs on PCR inhibition caused by two competitive primers allowed investigation of the mechanisms of AuNP-enhanced PCR accuracy. Finally, heat-treated salmon sperm DNA was used to evaluate whether AuNPs could eliminate the inhibitory PCR effect caused by DNA impurities. This study demonstrated that enhanced thermal conductivity by AuNPs was the main mechanism for increased PCR efficiency and specificity. AuNPs promoted efficient double-stranded DNA template unwinding and dissociation between mismatched primers, DNA fragments and template, and enhanced PCR efficiency and specificity simultaneously. Thus, this significant finding suggests the future use of AuNP-assisted PCR in different fields, especially in rapid clinical diagnosis and screening by PCR.

Precision Templating with DNA of a Virus-like Particle with Peptide Nanostructures

We report here the preparation of filamentous virus-like particles by the encapsulation of a linear or circular double-stranded DNA template with preassembled mushroom-shaped nanostructures having a positively charged domain. These nanostructures mimic the capsid proteins of natural filamentous viruses and are formed by self-assembly of coiled-coil peptides conjugated at opposite termini with cationic segments and poly(ethylene glycol) (PEG) chains. We found that a high molecular weight of PEG segments was critical for the formation of monodisperse and uniformly shaped filamentous complexes. It is proposed that electrostatic attachment of the nanostructures with sufficiently long PEG segments generates steric forces that increase the rigidity of the neutralized DNA template. This stiffening counterbalances the natural tendency of the DNA template to condense into toroids or buckle multiple times. The control achieved over both shape and dimensions of the particles offers a strategy to create one-dimensional supramolecular nanostructures of defined length containing nucleic acids.

Single-stranded DNA loops as fiducial markers for exploring DNA–protein interactions in single molecule imaging

A polymerase chain reaction (PCR) based method of adding a single-stranded DNA (ssDNA) hairpin loop to one end of linear double-stranded (ds) DNA templates was developed. The loop structure serves as a fiducial marker in single molecule imaging by atomic force microscopy (AFM) and can be applied to study DNA–protein interactions. The nucleic acid end-labels allow discrimination of the polarity of the DNA template in the AFM while limiting non-specific interactions which might occur from non-nucleic acid labels. Homo-polynucleotide ssDNA loops made up of 20 base-pairs (bp) for each of the four bases (A, T, G, C) were investigated to determine the effects of sequence on template labelling. The products were produced with high efficiency and high yield with the loop readily distinguished from the dsDNA template by height and diameter in the AFM. The application of the method to study DNA transcription was investigated by firing Escherichia Coli RNA polymerase (RNAP) from a λPR promoter in the direction of the loop-labelled end. The ssDNA loops captured elongating complexes of RNAP, arresting transcription and preventing dissociation. The dual role of the loop as a polarity marker and retainer of previously active RNAP will allow mechanisms of gene expression to be studied with single molecule sensitivity by AFM. This will enable insight into molecular interactions of RNAP on single DNA templates in convergent or tandem transcription configurations.

Double-strand DNA-templated synthesis of copper nanoclusters as novel fluorescence probe for label-free detection of biothiols

In this work, we report a simple and sensitive method for the detection of biothiols including glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), using fluorescent copper nanoclusters synthesized by a double-stranded DNA template (DNA-CuNCs) as a probe. The photoluminescence intensity of DNA-CuNCs was found to be quenched effectively with the increase in the concentration of biothiols due to the formation of nonfluorescent coordination complexes between the DNA-CuNCs and biothiols, whereas the fluorescence of DNA-CuNCs was not changed in the presence of other amino acids at a 10-fold higher concentration. Satisfactory detection limits and linear relationships for the detection of GSH, Cys and Hcy were obtained The resulting calibration plots exhibited good linear correlations in the range from 2.0 × 10−6 to 1.0 × 10−4 mol L−1 for Cys, 2.0 × 10−6 to 8.0 × 10−5 mol L−1 for GSH, and 5.0 × 10−6 to 2.0 × 10−4 mol L−1 for Hcy. The detection limits of Cys, GSH and Hcy were 2 μmol L−1, 2 μmol L−1 and 5 μmol L−1, respectively. In addition, the method was successfully applied in the detection of biothiols in human plasma samples.

Multiple RNA Polymerase Transactions on Single DNA Templates

We are studying the interactions of more than one RNA polymerase (RNAP) operating on single linear double-stranded DNA templates using imaging by atomic force microscopy (AFM) [1,2]. These templates include a short polynucleotide sequence of A, C, G or T forming a single-stranded end loop to discriminate polarity of the DNA molecules in the AFM [3]. Model templates include convergently and tandemly-aligned promoters for bacterial E. Coli RNAP.Measurement of the positions of the RNAP on the DNA before and after firing from the promoters allow us infer outcomes of “collision” events. The end-labelling ssDNA loop not only acts as a fiducial marker but also captures RNAPs that have fired from promoters directed towards and closest to the loop. This enables us to quantify different outcomes of these events between more than one RNAP operating in a convergent or tandem configuration. Interestingly, RNAPs which approach each other often stall before close contact is made, implying that transient DNA super-coiling between RNAP mediates their interactions. Furthermore, the distribution of outcomes for head-on (convergent transcription) and rear-end (tandem transcription) collision events are similar, suggesting that long range interactions mediated through the local DNA conformation serve to regulate RNAP activity and positioning on the templates. These experiments inform the regulation of genetic expression at the molecular level, including acting as simple models for nested genes.[1] Billingsley D.J., Bonass W.A, Crampton N., Kirkham J. and Thomson N.H. (2012) Physical Biology 9 021001.[2] Crampton N., Bonass W.A., Kirkham J., Rivetti C. and Thomson N.H. (2006) Nucleic Acids Research 34 (19) 5416-5425.[3] Billingsley D.J., Crampton N., Kirkham J., Thomson N.H., Bonass W.A (2012) Nucleic Acids Research 40 (13) e99.

Direct Visualization of the Movement of a Single T7 RNA Polymerase and Transcription on a DNA Nanostructure

Snapshots of transcription: Movement of a single molecule of T7 RNA polymerase (RNAP) along a double-stranded DNA (dsDNA) template attached to a DNA origami platform (green, see scheme) was recorded using high-speed AFM. A one kilobase template dsDNA containing the T7 promoter was used to record AFM images of transcription of the template dsDNA by RNAP and the resulting biotinylated RNA product was detected by streptavidin-labeling.