Double-stranded DNA Templates Research Articles

Electrochemical aptasensor based on DNA-templated copper nanoparticles and RecJf exonuclease-assisted target recycling for lipopolysaccharide detection.

An electrochemical aptasensor for detecting lipopolysaccharides (LPS) was fabricated based on DNA-templated copper nanoparticles (DNA-CuNPs) and RecJf exonuclease-assisted target recycling. The DNA-CuNPs were synthesized on a double-stranded DNA template generated through the hybridization of the LPS aptamer and its complementary chain (cDNA). In the absence of LPS, the CuNPs were synthesized on DNA double-strands, and a strong readout corresponding to the CuNPs was achieved at 0.10 V (vs. SCE). In the presence of LPS, the fabricated aptamer could detach from the DNA double-strand to form a complex with LPS, disrupting the template for the synthesis of CuNPs on the electrode. Meanwhile, RecJf exonuclease could hydrolyze the cDNA together with this single-stranded aptamer, releasing the LPS for the next round of aptamer binding, thereby enabling target recycling amplification. As a result, the electrochemical signal decreased and could be used to indicate the LPS content. The fabricated electrochemical aptasensor exhibited an extensive dynamic working range of 0.01 pg mL-1 to 100 ng mL-1, and its detection limit was 6.8 fg mL-1. The aptasensor also exhibited high selectivity and excellent reproducibility. Moreover, the proposed aptasensor could be used in practical applications for the detection of LPS in human serum samples.

Synthesis of silver nanoclusters using a double-stranded DNA template and its application for captopril detection

The purpose of this study was to explore the feasibility and mechanism of synthesizing silver nanoclusters (AgNCs) using a double-stranded DNA (dsDNA) template and to use the AgNCs as probes for captopril detection. The results showed that the binding of silver ions towards DNA bases deduced the destruction of the initial dsDNA structure, and AgNCs with decent quantum yield (43.54%) were obtained. The detection using these AgNCs probes showed good performance for captopril, with a linear range of 0.1–0.8 μg·mL−1 and a detection limit of 0.045 μg·mL−1. Recovery results in real samples of Apocynum venetum leaf tea and Ginkgo biloba leaf tablet demonstrated its good applicability. This work not only extended the understanding of DNA-AgNCs but also provided a fast and cost-effective tool for captopril detection.

Ratio of the Primers Used in Polymerase Chain Reaction-Stop Analysis Impacts the Resultant Banding Pattern.

G-quadruplex (G4), as a dynamic nucleic acid secondary structure, widely exists in organism genomes and plays regulatory roles in a variety of cellular functions. Polymerase chain reaction stop assay (PCR-Stop) is a simple, quick, and low-cost widely used method for detection of the binding between G4 and its binding compounds. Different from the common PCR approach, no double-stranded DNA template is needed in the PCR-Stop assay, in which the forward and reverse primers extend against each other in the presence of DNA polymerase to produce a single DNA product. However, unexpected results, such as two or more PCR products, are often generated, and the mechanism is unclear. We found that the ratio of pair primers significantly impacts the generation and components of PCR-Stop products, which is crucial for the interpretation of the experiment results.

Protein crystallization with DNA templates

Protein crystallization plays a significant role in three-dimensional structural analysis and protein purification. It is important to increase the crystallization efficiency, which is possible by adding heterogeneous templates in crystallization systems. DNA is biologically compatible and artificially designable polymer, which is easy to extract. In this study, single- and double-stranded DNA of precise sequences were designed and used as templates to promote protein crystallization of lysozyme and catalase. Influence of DNA, single-stranded DNA with 10, 20, 40 bases and double-stranded DNA with 10, 20, 40 base pairs, were investigated. The success rate of obtaining crystals of lysozyme and catalase in equal period was significantly improved with the addition of DNA comparing without templates added. Double-stranded DNA led to higher nucleation rate than that with single-stranded DNA. The promotion of nucleation was more obvious at low concentration of protein solution and with longer chain DNA templates. Crystal number and crystallization rate was enhanced with addition of long double-stranded DNA templates. All the results confirm that DNA is an effective polymer additive to enhance protein crystallization, especially for the application of the scarce protein crystallization.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

PASTE: The Way Forward for Large DNA Insertions.

PASTE: The Way Forward for Large DNA Insertions.

ALL-IN-ONE VIRUS-FREE MANUFACTURING PROCESS OF ALLOGENEIC CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS USING CRISPR/CAS9

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising therapeutic opportunity against blood cancers, leading to the six FDA-approved CAR T cell products. All commercially available products are produced following an autologous strategy, which represents a bottleneck for the widespread adoption of this technology due to the poor quality and quantity of patients’ T cells. Allogeneic T cells from healthy donors are an excellent alternative over the autologous ones, allowing the production of a ready-to-use therapy besides additional benefits, such as standardization of the final product, cost reduction through manufacturing scale-up, and the possibility of multiple genetic modifications. Safety concerns related to the use of cells from third-party donors can be easily eliminated through T cell receptor (TCR) knock-out with the demonstrated prevention of graft-versus-host disease development. Here we describe CRISPR/Cas9 editing for TRAC ( T Cell Receptor Alpha Constant )-targeted genomic integration of the CAR transgene and the consequent TCR knock-out in a fast and efficient virus-free manufacturing process. For the CRISPR/Cas9-mediated knock-in, we designed a plasmid containing the second-generation anti-CD19 CAR flanked by arms homologous to the desired cut site. This sequence was PCR amplified and purified, resulting in a highly concentrated double-stranded DNA (dsDNA) template for the homology-directed repair (HDR). Human T cells isolated from healthy donors were electroporated with the dsDNA-HDR templates (dsDNA-HDRT) and Cas9 ribonucleoproteins (RNPs) targeting the human TRAC locus. Cells were assayed on days 5 and 11 post-electroporation for CAR expression and killing efficiency, respectively. A mCherry transgene flanked by the same homology arms was also included as a control. Preliminary studies using the mCherry dsDNA-HDRT indicated a 1.2-fold improvement in the editing efficiency by adding the positively charged polymer poly-L-glutamic acid (40 μg) that binds to RNPs and reduces aggregates. Additionally, by culturing the electroporated cells with varying concentrations of an NHEJ inhibitor, we observed a 1.4 to 2-fold increase in mCherry knock-in, suggesting a favorable condition for the HDR. Based on the established optimized conditions, we electroporated T cells with the CAR dsDNA-HDRT (3.1 kb length, 6 μg) and got 88% of CAR+ cells five days after electroporation. The viability was 27% on day 3 and reached about 60% on day 7. On-target CAR gene integration was confirmed through an “in-out” PCR assay on the genomic DNA using primers specific to the TRAC locus and CAR transmembrane domain. We then analyzed the in vitro cytotoxicity of CAR T cells by coculturing them with GFP-expressing tumor cell lines. Complete growth inhibition of the RS4-11 cells (CD19 + ) was achieved at the 5:1, 10:1, and 50:1 effector-to-target ratios. For the K562 cells (CD19-), conversely, the killing activity was related to the T cells themselves since CAR expression did not further decrease the tumor cell number. These results demonstrate robust cytotoxic function and target specificity of the CAR T cell products. Combined, the data provide a proof-of-concept for the virus-free manufacturing process of allogeneic anti-CD19 CAR T cells, enabling CAR knock-in and TCR knock-out in an all-in-one strategy.

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays.

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between vertebrates and ticks and dramatically remodels its transcriptional profile to survive in disparate environments during infection. A focus of B. burgdorferi studies is to clearly understand how the bacteria responds to its environment through transcriptional changes. In vitro transcription assays allow for the basic mechanisms of transcriptional regulation to be biochemically dissected. Here, we present a detailed protocol describing B. burgdorferi RNA polymerase purification and storage, sigma factor purification, DNA template generation, and in vitro transcription assays. The protocol describes the use of RNA polymerase purified from B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC is a previously published strain harboring a 10XHis-tag on the rpoC gene encoding the largest subunit of the RNA polymerase. In vitro transcription assays consist of the RNA polymerase purified from strain 5A4-RpoC, a recombinant version of the housekeeping sigma factor RpoD, and a PCR-generated double-stranded DNA template. While the protein purification techniques and approaches to assembling in vitro transcription assays are conceptually well understood and relatively common, handling considerations for RNA polymerases often differ from organism to organism. The protocol presented here is designed for enzymatic studies on the B. burgdorferi RNA polymerase. The method can be adapted to test the role of transcription factors, promoters, and post-translational modifications on the activity of the RNA polymerase.

Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing.

Targeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive guanine-cytosine (GC) sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogrammed guide RNAs to direct CRISPR-Cas12a cleavage of double-stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7176 guides targeting 3.5 Mb of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 Mb. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for single nucleotide variants, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double-stranded DNA template or genome.

CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

A versatile fluorescent nanobeacon lighted by DNA-templated copper nanoparticles and the application in isothermal amplification detection

In this work, an environmentally-friendly and versatile nanobeacon was constructed by utilizing DNA-templated copper nanoparticles (CuNPs) as fluorescence signal source. As the key component of the nanobeacon, a hairpin DNA was designed to contain four segments: two segments for CuNPs template sequence, a target recognition segment and a blocking segment. At room temperature, the target recognition segment partly hybridizes with the blocking segment and thus prohibits the formation of double stranded DNA template, so that no CuNPs can be generated on the hairpin DNA. While a target is introduced, the specific binding of target with recognition sequence triggers off the conformational transformation of the hairpin DNA, which contributes to the formation of the CuNPs template. As a result, the in-situ generation of CuNPs gives birth to the fluorescence signal readout that can be used to identify the target. By reasonably varying the recognition sequence within hairpin DNA, a series of nanobeacons in response to corresponding targets, such as DNA, microRNA, thrombin, and ATP, were put forward with satisfactory sensitivity and selectivity. Moreover, this nanobeacon was also integrated with the strategy of enzyme-assisted target-recycling to realize signal amplification and ultrasensitive detection, which further demonstrated the versatility of the nanobeacon. This novel nanobeacon is expected to be a promising alternative to classical dye-labeled molecular beacon and provide new perspective on ultrasensitive fluorescence sensing.

TiO2-sensitized double-shell ZnCdS hollow nanospheres for photoelectrochemical immunoassay of carcinoembryonic antigen coupled with hybridization chain reaction-dependent Cu2+ quenching

A novel photoelectrochemical immunosensor was constructed to monitor carcinoembryonic antigen (CEA) based on hybridization chain reaction (HCR)-mediated in situ generation of copper nanoparticles (Cu NPs) and subsequent Cu2+-dependent quenching reaction, in which titanium dioxide nanoparticles-sensitized double-shell zinc cadmium sulfide hollow nanospheres (TiO2/DS-ZnCdS)-modified ITO electrode and anti-CEA antibody-modified 96-well plate served as biological recognition and signal detection platforms, respectively. The synergistic effect of TiO2 NPs and DS-ZnCdS hollow nanospheres contributed to the improvement of photoelectric conversion efficiency, and HCR-mediated signal cascade benefited the enhancement of detection sensitivity. In the presence of CEA, biotin-labelled anti-CEA antibodies were immobilized onto anti-CEA antibody-modified 96-well plate, and triggered HCR process to form long double stranded DNA, which could adsorb a large number of Cu2+ ions and then in situ form Cu NPs on double stranded DNA template by a facile reduction reaction. After acid treatment, the dissolved Cu2+ ions could significantly reduce the photocurrent response due to the generation of CuxS. Under optimal conditions, the immunosensor exhibited a desirable liner range of 1 pg mL−1 – 50 ng mL−1 and a low detection limit of 0.1 pg mL−1, as well as excellent selectivity and stability. Meanwhile, the recoveries of human serum sample analysis ranged from 96.8% to 103.6%, and the relative standard deviation was less than 7.40%, showing a good feasibility in early clinical diagnosis.

A Rad51-independent pathway promotes single-strand template repair in gene editing.

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

A simplified method to produce mRNAs and functional proteins from synthetic double-stranded DNA templates.

We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning.

Reconfigurable T-junction DNA Origami.

DNA self-assembly allows the construction of nanometre-scale structures and devices. Structures with thousands of unique components are routinely assembled in good yield. Experimental progress has been rapid, based largely on empirical design rules. Herein, we demonstrate a DNA origami technique designed as a model system with which to explore the mechanism of assembly. The origami fold is controlled through single-stranded loops embedded in a double-stranded DNA template and is programmed by a set of double-stranded linkers that specify pairwise interactions between loop sequences. Assembly is via T-junctions formed by hybridization of single-stranded overhangs on the linkers with the loops. The sequence of loops on the template and the set of interaction rules embodied in the linkers can be reconfigured with ease. We show that a set of just two interaction rules can be used to assemble simple T-junction origami motifs and that assembly can be performed at room temperature.

SinoDuplex: An Improved Duplex Sequencing Approach to Detect Low-frequency Variants in Plasma cfDNA Samples

Accurate detection of low frequency mutations from plasma cell-free DNA in blood using targeted next generation sequencing technology has shown promising benefits in clinical settings. Duplex sequencing technology is the most commonly used approach in liquid biopsies. Unique molecular identifiers are attached to each double-stranded DNA template, followed by production of low-error consensus sequences to detect low frequency variants. However, high sequencing costs have hindered application of this approach in clinical practice. Here, we have developed an improved duplex sequencing approach called SinoDuplex, which utilizes a pool of adapters containing pre-defined barcode sequences to generate far fewer barcode combinations than with random sequences, and implemented a novel computational analysis algorithm to generate duplex consensus sequences more precisely. SinoDuplex increased the output of duplex sequencing technology, making it more cost-effective. We evaluated our approach using reference standard samples and cell-free DNA samples from lung cancer patients. Our results showed that SinoDuplex has high sensitivity and specificity in detecting very low allele frequency mutations. The source code for SinoDuplex is freely available at https://github.com/SinOncology/sinoduplex.

Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.

The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.

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.

Biochemical methods to characterize RNA polymerase II elongation complexes.

Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.