dsDNA Template Research Articles

Chemical roadblocking of DNA transcription for nascent RNA display

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription "roadblocking" approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2'-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

Reprogramming Human T Cell Function and Specificity With Non–Viral Genome Targeting

TL Roth, C Puig–Saus, R Yu. Nature. 2018;559(7714):405–409 To develop and optimize nonviral CRISPR-Cas9 genome editing for primary T cells and apply this technique in part to correct a pathogenic mutation in a monogenic autoimmune disease. Using CRISPR-Cas9, primary human T cells from healthy donors and a family with monogenic autoimmune disease were engineered ex vivo along with other important proofs of concept. The CRISPR-Cas9 system was first described as a prokaryotic adaptive immune response, and it has since been widely used as a gene editing tool. CRISPR sequences identify homologous areas in the DNA, and Cas9 creates precise double-stranded breaks. Double-stranded breaks are repaired by either nonhomologous end joining or homology-directed repair (HDR). HDR is less error prone, and by using CRISPR-Cas9 with HDR templates, transgenes can be integrated into the targeted genome with precision. In this study, an electroporation method was optimized for ex vivo primary human T cells to allow for uptake of CRISPR-Cas9 ribonucleoprotein complexes and large (>1 kb) double-stranded DNA (dsDNA) templates for HDR. The technique allowed for optimized cell viability along with efficiency under …

Efficient Long-Range, Directional Energy Transfer through DNA-Templated Dye Aggregates.

The benzothiazole cyanine dye K21 forms dye aggregates on double-stranded DNA (dsDNA) templates. These aggregates exhibit a red-shifted absorption band, enhanced fluorescence emission, and an increased fluorescence lifetime, all indicating strong excitonic coupling among the dye molecules. K21 aggregate formation on dsDNA is only weakly sequence dependent, providing a flexible approach that is adaptable to many different DNA nanostructures. Donor (D)-bridge (B)-acceptor (A) complexes consisting of Alexa Fluor 350 as the donor, a 30 bp (9.7 nm) DNA templated K21 aggregate as the bridge, and Alexa Fluor 555 as the acceptor show an overall donor to acceptor energy transfer efficiency of ∼60%, with the loss of excitation energy being almost exclusively at the donor-bridge junction (63%). There was almost no excitation energy loss due to transfer through the aggregate bridge, and the transfer efficiency from the aggregate to the acceptor was about 96%. By comparing the energy transfer in templated aggregates at several lengths up to 32 nm, the loss of energy per nanometer through the K21 aggregate bridge was determined to be <1%, suggesting that it should be possible to construct structures that use much longer energy transfer "wires" for light-harvesting applications in photonic systems.

One-Pot Production of RNA Nanoparticles via Automated Processing and Self-Assembly.

From the original sequencing of the human genome, it was found that about 98.5% of the genome did not code for proteins. Subsequent studies have now revealed that a much larger portion of the genome is related to short or long noncoding RNAs that regulate cellular activities. In addition to the milestones of chemical and protein drugs, it has been proposed that RNA drugs or drugs targeting RNA will become the third milestone in drug development ( Shu , Y. ; Adv. Drug Deliv. Rev. 2014 , 66 , 74 . ). Currently, the yield and cost for RNA nanoparticle or RNA drug production requires improvement in order to advance the RNA field in both research and clinical translation by reducing the multiple tedious manufacturing steps. For example, with 98.5% incorporation efficiency of chemical synthesis of a 100 nucleotide RNA strand, RNA oligos will result with 78% contamination of aborted byproducts. Thus, RNA nanotechnology is one of the remedies, because large RNA can be assembled from small RNA fragments via bottom-up self-assembly. Here we report the one-pot production of RNA nanoparticles via automated processing and self-assembly. The continuous production of RNA by rolling circle transcription (RCT) using a circular dsDNA template is coupled with self-cleaving ribozymes encoded in the concatemeric RNA transcripts. Production was monitored in real-time. Automatic production of RNA fragments enabled their assembly either in situ or via one-pot co-transcription to obtain RNA nanoparticles of desired motifs and functionalities from bottom-up assembly of multiple RNA fragments. In combination with the RNA nanoparticle construction process, a purification method using a large-scale electrophoresis column was also developed.

Detection of micrococcal nuclease for identifying Staphylococcus aureus based on DNA templated fluorescent copper nanoclusters.

Micrococcal nuclease (MNase) is a naturally-secreted nucleic acid degrading enzyme with important role in the spread of the bacteria in an infected host. The content of MNase can be used to estimate the pathogenicity of Staphylococcus aureus (S. aureus). A fluorometricmethod is described here for determination of the activity of MNase and for identification of S. aureus using DNA templated fluorescent copper nanoclusters (CuNC). A double-stranded DNA (dsDNA) with AT-rich regions and protruding 3'-termini was identified as a high-selectivity substrate for MNase and as a template for CuNC. In the absence of MNase, the long AT-rich dsDNA templates the formation of CuNC that display bright yellow fluorescence, with excitation/emission peaks at 340/570nm. However, the substrates are enzymatically digested to mononucleotides or short-oligonucleotide fragments, which fail to synthesize fluorescent CuNC. The method works in the 1.0 × 10-3 - 5.0 × 10-2UmL-1 MNase activity range, has a 1.0mUmL-1 detection limit, and is highly selective over other exonucleases. The assay was successfully applied to the detection of MNase secreted by S. aureus and to the identification of S. aureus. Graphical abstract A smart dsDNA, with AT-rich regions and 3'-protruding termini, is screened as the high-selectivity substratefor MNase and template for the formation of copper nanoclusters (CuNC). A method is described for determination of the activity of MNase and for identification of S. aureus via smart DNA templatedformation of fluorescent CuNCs.

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.

Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters.

A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3'-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07UmL-1 to 15UmL-1 and a detection limit of 0.06UmL-1. Graphical abstract The sequence-dependent fluorescence of DNA-templated copper nanoclusters, which are in-situ synthesized through the reduction of CuSO4 by ascorbate (Vc) in the presence of dsDNA template, is utilized to obtain the method for sensitive detection of T4 PNKP activity with near-zero background.

Reproducible Enhancement of Fluorescence by Bimetal Mediated Surface Plasmon Coupled Emission for Highly Sensitive Quantitative Diagnosis of Double-Stranded DNA.

Plasmonic enhancement of fluorescence from SYBR Green I conjugated with a double-stranded DNA (dsDNA) amplicon is demonstrated on polymerase chain reaction (PCR) products. Theoretical computation leads to use of the bimetallic (Au 2 nm-Ag 50 nm) surface plasmons due to larger local fields (higher quality factors) than monometallic (Ag or Au) ones at both dye excitation and emission wavelengths simultaneously, optimizing fluorescence enhancement with surface plasmon coupled emission (SPCE). Two kinds of reverse Kretschmann configurations are used, which favor, in signal-to-noise ratio, a fluorescence assay that uses optically dense buffer such as blood plasma. The fluorescence enhancement (12.9 fold at maximum) with remarkably high reproducibility (coefficient of variation (CV) < 1%) is experimentally demonstrated. This facilitates credible quantitation of enhanced fluorescence, however unlikely to obtain by localized surface plasmons. The plasmon-induced optical gain of 46 dB due to SPCE-active dye molecules is also estimated. The fluorescence enhancement technologies with PCR enables LOD of the dsDNA template concentration of ≈400 fg µL-1 (CV < 1%), the lowest ever reported in DNA fluorescence assay to date. SPCE also reduces photobleaching significantly. These technologies can be extended for a highly reproducible and sufficiently sensitive fluorescence assay with small volumes of analytes in multiplexed diagnostics.

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.

Fluorescent DNA-Templated Silver Nanoclusters from Silver(I)-Mediated Base Pairs.

Silver nanoclusters (AgNCs) stabilized by double-stranded DNA (dsDNA) are of special interest because the duplex structures provide rigidity and chirality that can be transferred to the metal clusters. This work reports fluorescent AgNCs obtained from dsDNA templates containing artificial ligand-derived nucleobases. They are compared with fluorescent AgNCs obtained from DNA templates containing C:C mismatches (C, cytosine). Towards this end, the new metal-mediated Im-AgI -C base pair composed of imidazole (Im) and cytosine was introduced into dsDNA and reduced to form AgNCs. The clusters were characterized by UV/Vis, fluorescence and circular dichroism spectroscopy. The AgNCs form chiral aggregates with dsDNA. Their optical properties are highly sequence-dependent. As a result, a simple method to detect a cytosine insertion into a run of cytosine residues in a duplex is proposed using the human CDH1 gene as an example.

3D contour fluorescence spectroscopy with Brus model: Determination of size and band gap of double stranded DNA templated silver nanoclusters

Here, we propose a new synthetic methodology for silver nanocluster preparation by using a double stranded-DNA (ds-DNA) template which no one has reported yet. A new calculative method was formulated to determine the size of the nanocluster and their band gaps by using steady state 3D contour fluorescence technique with Brus model. Generally, the structure and size of the nanoclusters determine by using High Resolution Transmission Electron Microscopy (HR-TEM). Before imaging the samples by using HR-TEM, they are introduced to drying process which causes aggregation and forms bigger polycrystalline particles. It takes long time duration and expensive methodology. In this current methodology, we found out the size and band gap of the nanocluster in the liquid form without any polycrystalline aggregation for which 3D contour fluorescence technique was used as an alternative approach to the HR-TEM method.

A novel silver nanocluster in situ synthesized as versatile probe for electrochemiluminescence and electrochemical detection of thrombin by multiple signal amplification strategy

In this work, a novel silver nanoclusters (AgNCs) were in situ synthesized and used as versatile electrochemiluminescence (ECL) and electrochemical (EL) signal probes for thrombin detection by using DNAzyme-assisted target recycling and hybridization chain reaction (HCR) multiple amplification strategy. The presence of target thrombin firstly opened the hairpin DNA, followed by DNAzyme-catalytic recycling cleavage of excess substrates, which could generate large number of substrate fragments (s1). Then these s1 fragments were captured by SH–DNA on the Au nanoparticle-modified electrode, which further triggered the subsequent HCR of the hairpin DNA probes (H1 and H2) to form the long dsDNA. The numerous AgNCs were thus in situ synthesized by incubation the dsDNA template (with cytosine-rich loop)-modified electrode in solution with AgNO3 and sodium borohydride. By integrating the DNAzyme recycling and HCR dual amplification strategy, the amount of AgNCs is dramatically enhanced, leading to substantially amplified ECL and electrochemical signals for sensitive thrombin detection. Importantly, this design introduces the novel AgNCs into versatile ECL and EL bioassays by multiple amplification strategy, thus it is promising to provide a highly sensitive platform for various target biomolecules.

Design and Preparation of Aptamer-siRNA Chimeras (AsiCs) for Targeted Cancer Therapy.

Conjugation of cell-internalizing DNA or RNA aptamers to tumor-suppressing siRNAs represents a novel promising approach for cancer therapy. Here we describe how to employ RNA aptamers that bind to cell surface receptors as carriers for cell-targeted siRNA (or miRNA) delivery. This protocol was optimized to improve the efficiency of the aptamer-siRNA/miRNA conjugates and facilitate its implementation in most molecular biology labs. The single working steps include (1) outlining the optimal sequences of the RNA strands bearing the aptamer and siRNA sequence, for which further (2) a dsDNA template is synthesized and then (3) transcribed into an RNA that will be (4) folded and annealed to a chemically synthesized siRNA complementary strand. Moreover, we reference recent examples and advances in the aptamer delivery field.

Cotranscriptional Production of Chemically Modified RNA Nanoparticles.

RNA nanoparticles consisting of multiple RNA strands of different sequences forming various three-dimensional structures emerge as promising carriers of siRNAs, RNA aptamers, and ribozymes. In vitro transcription of a mixture of dsDNA templates encoding all the subunits of the RNA nanoparticle may result in cotranscriptional self-assembly of the nanoparticle. Based on our experience with production of RNA nanorings, RNA nanocubes, and RNA three-way junctions, we propose a strategy for optimization of the cotranscriptional production of chemically modified ribonuclease-resistant RNA nanoparticles.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses.

BackgroundViruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).MethodsHere we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.ResultsMock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample.DiscussionTogether these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5' to 3' translocase on single-stranded DNA.

A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence

PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5' end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.

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.

Label-free electrochemical detection of methyltransferase activity and inhibitor screening based on endonuclease HpaII and the deposition of polyaniline

Detection of DNA methylation and methyltransferase (MTase) activity are important in determining human cancer because aberrant methylation was linked to cancer initiation and progression. In this work, we proposed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on methylation sensitive restriction endonuclease HpaII and the deposition of polyaniline (PANI) catalyzed by HRP-mimicking DNAzyme. In the presence of methylated DNA, HRP-mimicking DNAzyme catalyzed the polymerization of aniline on the dsDNA template, producing huge DPV current. In the presence of non-methylated DNA, dsDNA are cleaved and digested by HpaII and exonuclease III, as a result, no PANI are deposited. This method can be used to determine DNA methylation at the site of CpG. It exhibits a wide linear response toward M.SssI MTase activity in the range of 0.5-0.6 U mL(-1) with the detection limit of 0.12 U mL(-1). G-rich DNA forms HRP mimicking DNAzyme, which avoids complex labeling procedures and is robust. The method is simple, reliable, sensitive and specific, which has been successfully applied in human serum samples and been used to screen the inhibitors. Thus, the proposed method may be a potential and powerful tool for clinical diagnosis and drug development in the future.