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Exosomes are emerging as one of the most promising biomarkers for early disease diagnosis and prognosis. The significant challenges facing the available methods include improving the detection specificity and sensitivity in complex biological samples. Herein, a fluorescence assay was established based on a combination of immunomagnetic separation and a two-step signal amplification strategy for direct isolation and subsequent detection of exosomes. First, immunomagnetic beads capture and enrich the exosomes via antibody-antigen reactions. Second, bivalent cholesterol (BC) anchors spontaneously insert into the lipid bilayer of bead-captured exosomes, forming a "one to many" amplification effect. The simultaneous recognition of the surface protein and the lipid bilayer structure of the exosome significantly eliminates the interference risk from free proteins. The detection of exosomes converts to the detection of BC-anchors. Finally, the sticky end of the BC-anchor acts as the initiator to trigger the enzyme-free DNA circuits for secondary signal amplification. Under the optimal conditions, highly sensitive and selective detection of exosomes was achieved ranging from 5.5 × 103 to 1.1 × 107 particles/μL with a limit of detection of 1.29 × 103 particles/μL. Moreover, this method allows the isolation and quantitative analysis of exosomes in several biological fluids with satisfactory recovery rates (92.25-106.8%). Thus, this approach provides a sensitive, anti-interference platform for isolating and detecting exosomes.

The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.

DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (k(pol)), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme–substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates. [Image: see text]

BAC library has always been an important genomic source for preserving the genetic material of an organism for sequencing, developing physical map as well as addressing the novel evolutionary biology questions. Here, the end sequences of BAC clones were generated using a set of primers and mapped on the genome scaffolds of C. magur using bioinformatics tool. Numbers of genes present on the clones were identified using scaffolds’ annotation information. Further, the clone insert DNA was labeled through nick translation using tetra methyl rhodamine-5-dUTP (Pool A contained 6 clones possessing 36 genes mapped on scaffold A) and fluorescein 12-dUTP (Pool B contained 4 clones possessing 15 genes mapped on scaffold B) fluorophores for probe construction and was subsequently hybridized on metaphase chromosomes using FISH. The BAC-FISH revealed the mapping of red color probe on 19th sub-telocentric chromosome and green color on 14th sub-metacentric chromosome. These findings will be useful in understanding the chromosomes organization and evolution of genomic structure as well as function of the species in comparison to its closely related and well studies species, such as channel catfish and zebrafish.

CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader–repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3′ end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader–repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1–Cas2 varied, with G1 stringently requiring a 5-nucleotide 3′ overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein–DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1–Cas2 toolset for inserting small DNAs into diverse DNA targets.

CRISPR–Cas9 methods have been applied to generate random insertions and deletions, large deletions, targeted insertions or replacements of short sequences, and precise base changes in plants1–7. However, versatile methods for targeted insertion or replacement of long sequences and genes, which are needed for functional genomics studies and trait improvement in crops, are few and largely depend on the use of selection markers8–11. Building on methods developed in mammalian cells12, we used chemically modified donor DNA and CRISPR–Cas9 to insert sequences of up to 2,049 base pairs (bp), including enhancers and promoters, into the rice genome at an efficiency of 25%. We also report a method for gene replacement that relies on homology-directed repair, chemically modified donor DNA and the presence of tandem repeats at target sites, achieving replacement with up to 130-bp sequences at 6.1% efficiency. Rice genes are replaced using chemically modified donor DNA and CRISPR targeting.

Bacteria have evolved diverse mechanisms to fend off predation by bacteriophages. We previously identified the Dnd system, which uses DndABCDE to insert sulfur into the DNA backbone as a double-stranded phosphorothioate (PT) modification, and DndFGH, a restriction component. Here, we describe an unusual SspABCD-SspE PT system in Vibrio cyclitrophicus, Escherichia coli and Streptomyces yokosukanensis, which has distinct genetic organization, biochemical functions and phenotypic behaviour. SspABCD confers single-stranded and high-frequency PTs with SspB acting as a nickase and possibly introducing nicks to facilitate sulfur incorporation. Strikingly, SspABCD coupled with SspE provides protection against phages in unusual ways: (1) SspE senses sequence-specific PTs by virtue of its PT-stimulated NTPase activity to exert its anti-phage activity, and (2) SspE inhibits phage propagation by introducing nicking damage to impair phage DNA replication. These results not only expand our knowledge about the diversity and functions of DNA PT modification but also enhance our understanding of the known arsenal of defence systems.

Recently newer synthetic DNA vaccines have been rapidly advanced to clinical study and have demonstrated an impressive degree of immune potency and tolerability. Improvements in DNA delivery over prior needle and syringe approaches include jet delivery, gene gun delivery, among others. Among the most effective of these new delivery methods, advanced electroporation (EP), combined with other advances, induces robust humoral and cellular immunity in both preventative as well as therapeutic studies. Advancements in the design of the DNA inserts include leader sequence changes, RNA and codon optimizations, improved insert designs, increased concentrations of DNA, and skin delivery, appear to complement newer delivery strategies. These advances also provide a framework for the in vivo production of synthetic DNA biologics. In this review, we focus on recent studies of synthetic DNA vaccines in the clinic for the prevention or treatment of infectious diseases with a focus on adaptive electroporation for delivery, and briefly summarize novel preclinical data advancing the in vivo delivery of DNA-encoded antibody-like biologics.

The CRISPR-associated transposase system enables site-specific DNA integration on the genome independent of homologous recombination. Previous studies have demonstrated that the type V-K CRISPR-associated Tn7-like transposase system from Scytonema hofmanni and the type I-F system from Vibrio cholerae have strong target immunity like Tn7, and therefore two or more copies of the donor DNA would not be inserted into the same target location in theory. In this paper, we report that the type I-F system can insert multiple donor copies into one site, which was identified and confirmed by single-strain identification and high-throughput sequencing. This result is beneficial for our application of multicopy chromosomal integration by CRISPR-associated transposases, allowing more donor insertions into the chromosome. This unexpected result shows that the target immunity mechanism of this system has not been fully understood. Attention should be paid to the possibility of multiple insertions and their effects in related research.

We developed a simplified, highly efficient Gateway reaction that recombines target DNA to expression (destination) plasmids in vivo and subsequently conjugates the final vector into a recipient strain, all in a single step. This recipient strain does not need to contain any selective marker and can be freely chosen as long as it is sensitive to ccdB counterselection and can be targeted by the RP4α conjugation system. Our protocol is simple, robust, and cost effective. It works in 96-well plate format and performs across a range of temperatures. We designed modular, minimal destination vectors containing a modified Gateway insert to ease vector design by providing locations for insertion of tags, promoters, or conjugations. To demonstrate the utility of our system, we created destination vectors with split adenylate cyclase tags for bacterial two-hybrid (B2H) studies and screened a library of diguanylate cyclases for protein-protein interactions in a single step.

Caenorhabditis elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination-mediated cassette exchange (RCME). This strain is unc-119(−) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual-color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

SummaryGenetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell’s genome, allowing for the later reconstruction of cellular experiences using high-throughput DNA sequencing1. Existing DNA recorders have achieved high-information recording2–14, durable recording3,5–10,13,15–18, prolonged recording over multiple timescales3,5,8,10, multiplexed recording of several user-selected signals5–8,18, and temporally resolved signal recording5–8,18, but not all at the same time. We present a DNA recorder called peCHYRON (prime editing19 Cell HistorY Recording by Ordered iNsertion) that does. In peCHYRON, prime editor guide RNAs19 (pegRNAs) insert a variable triplet DNA sequence alongside a constant propagation sequence that deactivates the previous and activates the next step of insertion. This process results in the sequential accumulation of regularly spaced insertion mutations at a synthetic locus. Accumulated insertions are permanent throughout editing because peCHYRON uses a prime editor that avoids cutting both DNA strands, which risks deletions. Editing continues indefinitely because each insertion adds the complete sequence needed to initiate the next step. Constitutively expressed pegRNAs generate insertion patterns that support straightforward reconstruction of cell lineage relationships. Pulsed expression of different pegRNAs enables the reconstruction of pulse sequences, which may be coupled to biological stimuli for temporally-resolved multiplexed event recording.

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA-repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2-5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.

Pore-forming proteins are an agent for attack or defense in various organisms, and its cytolytic activity has medical potential in cancer therapy. Despite recent advances in mimicking these proteins by amphipathic DNA nanopores, it remains inefficient to incorporate them into lipid bilayers. Here, we present the development of vesicular DNA nanopores that can controllably open a lipid membrane. Different from previously reported DNA nanopores that randomly insert into the planar bilayers, we design on-command fusogenic liposome-incorporated transmembrane DNA nanopores (FLIPs) that bypass the direct insertion process. By steric deshielding of fusogenic liposomal supports under low pH conditions, the embedded FLIPs are transferred and perforate lipid bilayers. We find that FLIPs depolarize the plasma membrane and thereby induce pyroptosis-like cell death. We further demonstrate the use of FLIPs to inhibit tumor growth in murine tumor models, which provides a new route to cancer nanotherapy.

Bridged oligonucleotides that contain nonnucleotide inserts, i.e., diethylene glycol phosphate residues (DEG insert) have been studied. These oligonucleotide derivatives were shown to smooth the melting temperatures of their DNA duplexes of various nucleotide composition. The DEG insert has been shown to have almost no effect on the sequence specificity of modified oligonucleotides and regulate only their hybridization properties. It has been demonstrated that bridged oligonucleotides immobilized on microparticles can be successfully used to specifically and efficiently reveal DNA templates during the parallel solid-phase hybridization analysis. The use of bridged oligonucleotides along with DNA-dependent enzymes (Taq DNA polymerase and T4 DNA ligase) in this analysis has led to pronounced discrimination of wrong DNA molecules due to the covalent attachment of the label to the carrier, thus increasing the reliability of the hybridization analysis.

PURPOSE: Single-stranded DNA (ssDNA) library has shown to enrich shorter and more degraded DNA fragments than double-stranded DNA (dsDNA) library. In this study, we evaluated whether ssDNA libraries capture more circulating tumor DNAs (ctDNAs) in plasma cell-free DNA (cfDNA). MATERIALS AND METHODS: We prepared dsDNA, ssDNA and pure-ssDNA (capture the pre-existing ssDNA) libraries using ten plasma cfDNA samples. After low-pass whole genome sequencing, we calculated duplicate rate to estimate library complexity and compared the library insert sizes between different library methods. Finally, we estimated ctDNA content and plasma genomic abnormality (PGA) score, an indicator of ctDNA burden. RESULTS: 27 libraries were prepared and sequenced from the ten cfDNA samples. Duplicate rate in the ssDNA and pure-ssDNA libraries was significantly lower than dsDNA libraries (p< 0.001 and p< 0.01, respectively). ctDNA content and PGA scores were consistently higher in ssDNA and pure-ssDNA libraries than in matched dsDNA libraries (p< 0.005). The higher ctDNA content in ssDNA libraries was associated with smaller library insert size. CONCLUSIONS: ssDNA libraries preserve more diversity and capture more ctDNA than dsDNA libraries. The ssDNA library method is preferred when performing genomic analysis of ctDNA.

The existing molecular cloning methods are often limited by the availability of suitable restriction sites. It is still a challenge for simultaneous cloning of multiple fragments into different sites of a single vector. Here we developed a novel method named improved overlap extension PCR (IOEP) for restriction enzyme independent cloning of large fragments. The addition of primers enables IOEP to exponentially amplify the overlap extension product, thus greatly improves the amplification efficiency of large fragments. Moreover, coupled with the benefit of T4 DNA polymerase to improve cloning efficiency, our method can be used to simultaneously insert, delete and replace multiple DNA fragments at different sites.

Extraction of high-quality genomic DNA is an important requisite for a number of molecular- and genome-based studies. Isolation of good quantity and quality of genomic DNA from plant materials is a major concern for molecular biologists as many of them contain large amount of polysaccharides, phenolics and secondary metabolites. The presence of polyphenols and polysaccharides in plants results in a brown–dark brown-coloured, contaminated DNA which is sticky and viscous in nature. In our present study, we performed DNA isolation from two plants (tomato and chilli) using Dellaporta, modified CTAB, phenanthroline-based, CaCl2-based DNA extraction protocols (with and without high-salt Tris-EDTA buffer treatment). For chilli plants, Dellaporta method with high-salt TE buffer treatment was found to be better than other methods, and for tomato plants modified CTAB and phenanthroline-based methods without high-salt TE treatment were better. To further confirm the applicability, total DNA was isolated from 99 chilli samples using Dellaporta method with high-salt TE treatment. The plants were then screened for geminivirus and 49 were found to be positive. Furthermore, one of the PCR products of Deng primer was cloned and analysis of the clones showed the presence of insert, thus confirming the applicability of the DNA extraction method for detection of virus, PCR and cloning.

The high-throughput capacities of the Illumina sequencing platforms and the possibility to label samples individually have encouraged wide use of sample multiplexing. However, this practice results in read misassignment (usually

Despite the rapid development of modern methods of cancer treatments, chemotherapy is still one of the most important, and sometimes, the only one method of therapy. The development of cancer treatment methods indicates that search of the new, more effective and safer medicines are necessary. Treatment using common anticancer drugs is associated with their interaction with DNA. Further research on the binding of metal ions and their complexes to DNA resulted from their potential applications as new therapeutic and diagnostic agents. It is well known that mutations in DNA play a significant role in the formation of tumors, however not all of these processes are known. The recognition of binding lanthanide(III) ions or lanthanide(III) complexes to DNA and cleavage DNA via lanthanide(III) complexes are important to understand unknown mutations that lead to cancer and treatment of this disease. The scientists have been undertaking various attempts to interfere the human DNA in order to overcome genetic diseases. Gene therapy could be considered as a kind of scissors that allow to cleavage the DNA in the proper place and insert the corrective gene. Lanthanide(III) ions and their complexes, due to their unique properties, could be widely used as luminescent probes for bioassays and as reagents in optical and magnetic resonance imaging. The luminescence properties of these compounds also have been adopted in medicinal diagnosis. This review will summarize the role of lanthanide(III) ions and their compounds in recent studies on the use of lanthanide(III) complexes to bind, cleavage of DNA also in diagnosis, and monitoring the treatment of cancer disease.

Antimicrobial peptides of mastatopara-S (M-S), thanatin, and ponericin W1(P W1) were able to disrupt the membrane integrity and alter the morphology of the hyphae of Geotrichum citri-aurantii and then reduced the sour rot of citrus fruit. In order to understand the mechanisms of thanatin, P W1 and M-S other than membrane disruption, the interaction betwixt the peptides and G. citri-aurantii DNA were investigated in this research. The laser confocal microscopy found that P W1, thanatin, and M-S could penetrate the cell membrane. Gel retardation assay demonstrated that P W1, thanatin, and M-S could bind to the G. citri-aurantii genomic DNA in vitro. UV-visible spectra and fluorescence spectra analysis further confirmed that the peptides can bind to the DNA, and then insert into the base pairs in the DNA helix, followed by wrecking the double-helix structure. In addition, M-S, thanatin, and P W1 can suppress the synthesis of DNA and RNA of G. citri-aurantii.

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

HiSeq DNA pair-end library construction is used for DNA sequencing onIllumina HiSeq 2500/HiSeq 4000/HiSeq X Ten/ and NovaSeq sequencers. The insert size of the DNA fragments can be 300bp, 350bp and 500bp.

Knock-in of large transgenes by Cas9-mediated homology-directed repair (HDR) is an extremely inefficient process. Although the use of single-stranded oligonucleotides (ssODN) as an HDR donor has improved the integration of smaller transgenes, they do not support efficient insertion of large DNA sequences. In an effort to gain insights into the mechanism(s) governing the HDR-mediated integration of larger transgenes and to improve the technology, we conducted knock-in experiments targeting the human EMX1 locus and applied rigorous genomic PCR analyses in the human HEK293 cell line. This exercise revealed an unexpected molecular complication arising from the transgene HDR being initiated at the single homology arm and the subsequent genomic integration of plasmid backbone sequences. To pivot around this problem, we devised a novel PCR-constructed template containing blocked long 3' single-stranded overhangs (BL3SSO) that greatly improved the efficiency of bona fide Cas9-stimulated HDR at the EMX1 locus. We further refined BL3SSO technology and successfully used it to insert GFP transgenes into two important interferon-stimulated genes (ISGs) loci, Viperin/RSAD2, and ISG15. This study demonstrates the utility of the BL3SSO platform for inserting long DNA sequences into both constitutive and inducible endogenous loci to generate novel human cell lines for the study of important biological processes.

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe ‘glonads’, a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

The DNA mismatch repair (MMR) factor Mlh1–Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1–Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker's yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1–Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1–Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

During DNA replication, the deubiquitinating enzyme USP1 limits the recruitment of translesion polymerases by removing ubiquitin marks from PCNA to allow specific regulation of the translesion synthesis (TLS) pathway. USP1 activity depends on an allosteric activator, UAF1, and this is tightly controlled. In comparison to paralogs USP12 and USP46, USP1 contains three defined inserts and lacks the second WDR20‐mediated activation step. Here we show how inserts L1 and L3 together limit intrinsic USP1 activity and how this is relieved by UAF1. Intriguingly, insert L1 also conveys substrate‐dependent increase in USP1 activity through DNA and PCNA interactions, in a process that is independent of UAF1‐mediated activation. This study establishes insert L1 as an important regulatory hub within USP1 necessary for both substrate‐mediated activity enhancement and allosteric activation upon UAF1 binding., Insert L1 of USP1 plays multiples roles in regulating USP1 activity. It contributes to inhibition that is relieved upon UAF1 binding and is important for interaction with DNA loaded PCNA.

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilized across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites. Two of the most commonly used mutant sites are named lox66 and lox71, which recombine to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardized naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya

Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (

Synthetic genomics-driven dematerialization of genetic resources facilitates flexible hypothesis testing and rapid product development. Biological sequences have compositional biases, which, I reasoned, could be exploited for engineering of enhanced synthetic genomics systems. In proof-of-concept assays reported herein, the abundance of random oligonucleotides in viral genomic components was analyzed and used for the rational design of a synthetic genomics framework with plant virome capacity (SynViP). Type IIS endonucleases with low abundance in the plant virome, as well as Golden Gate and No See’m principles were combined with DNA chemical synthesis for seamless viral clone assembly by one-step digestion-ligation. The framework described does not require subcloning steps, is insensitive to insert terminal sequences, and was used with linear and circular DNA molecules. Based on a digital template, DNA fragments were chemically synthesized and assembled by one-step cloning to yield a scar-free infectious clone of a plant virus suitable for Agrobacterium-mediated delivery. SynViP allowed rescue of a genuine virus without biological material, and has the potential to greatly accelerate biological characterization and engineering of plant viruses as well as derived biotechnological tools. Finally, computational identification of compositional biases in biological sequences might become a common standard to aid scalable biosystems design and engineering.

C. elegans benefits from a large set of tools for genome manipulation. Yet, the insertion of large DNA constructs and the generation of inversions is still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that also serves as a landing pad for integration of transgenes by recombination mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations are relatively easy to obtain for insert sizes up to ~15 kb. Taking advantage of this integration method we establish a dual color fluorescent operon reporter system to study post-transcriptional regulation of mRNA. Last we show that large chromosomal segments can be inverted using phiC31 integrase. Thus the phiC31 integrase system should be a useful addition to the C. elegans toolkit.

We describe a rapid and reusable biophysical method to assay COVID-19 and other pathogens. The method uses fluorescent sensors (i.e. molecular beacons) designed to detect COVID-19 RNA or any RNA of interest, concurrent with an internal control without the need for amplification. The molecular beacons are stem-loop structures in which a ∼10 nucleotide loop region has the complementary sequence of a region of the target RNA, and a fluorophore and quencher are placed on the 5’ and 3’ ends of the stem. The energy of hybridization of the loop with its target is designed to be greater than the hybridization energy of the energy of the stem so that when the beacon encounters its target RNA, the structure opens resulting in dequenching of the fluorophore. Here, we designed a COVID-19 beacon that is completely quenched in its native form and undergoes a 50-fold increase in fluorescence when exposed to nanomolar amounts of synthetic viral oligonucleotide. No changes in intensity are seen when control RNA is added. A control beacon to a human GAPDH RNA, chosen for its high levels in saliva, behaved similar to the COVID-19 beacon. This increase in fluorescence with beacon opening can be completely reversed upon addition of single stranded DNA complementary to COVID-19 beacon loop region. Beacons can be attached to an insert matrix allowing their use in concentrated form and can be made from morphilino oligonucleotides that are resistant to RNases. We present an analysis of the parameters that will allow the development of test strips to detect virus in aerosol, body fluids and community waste.Statement of significanceA platform for reusable and rapid detection of COVID-19 RNA and other pathogenic RNAs without the need for amplification or sophisticated instrumentation in a complex environment is described.

Gene subcloning, a process in which the nucleotide sequence of interest is excised from on plasmid and inserted into another, seems to be an easy task to done. However, not all subcloning attempts are successful, even when the insert sequence and the double digested target plasmid are successfully purified form agarose gel and thought to be ready for subsequent ligation. In the current study we introduce a reliable, easy, and time consuming method for gene subcloning and also truncation. The stages are all carried out in a single microtube without any running on a gel, making it possible to accomplish a successful gene subcloning or truncation even with low concentrations of DNA molecules. Summarily, subcloning is achieved by mixing the plasmids of interest in a microtube and subjecting to restriction enzymes whose restriction sites flank the segment that is going to be subcloned. Digestion mixture is precipitated in the same microtube using isopropanol and the resultant DNA molecules are allowed to take part in a ligation reaction. The recombinant plasmids of interest are screened by colony PCR. Truncation is achieved by double- digestion of the plasmid of interest using a restriction enzyme whose restriction site flanks the segment that is going to be cut out.

DNA-assisted identification of historical remains requires the genetic analysis of highly degraded DNA, along with a comparison to DNA from known relatives. This can be achieved by targeting single nucleotide polymorphisms (SNPs) using a hybridization capture and next-generation sequencing approach suitable for degraded skeletal samples. In the present study, two SNP capture panels were designed to target similar to 25,000 (25 K) and similar to 95,000 (95 K) nuclear SNPs, respectively, to enable distant kinship estimation (up to 4th degree relatives). Low-coverage SNP data were successfully recovered from 14 skeletal elements 75 years postmortem using an Illumina MiSeq benchtop sequencer. All samples contained degraded DNA but were of varying quality with mean fragment lengths ranging from 32 bp to 170 bp across the 14 samples. SNP comparison with DNA from known family references was performed in the Parabon Fx Forensic Analysis Platform, which utilizes a likelihood approach for kinship prediction that was optimized for low-coverage sequencing data with cytosine deamination. The 25 K panel produced 15,000 SNPs on average, which allowed for accurate kinship prediction with strong statistical support in 16 of the 21 pairwise comparisons. The 95 K panel increased the average SNPs to 42,000 and resulted in an additional accurate kinship prediction with strong statistical support (17 of 21 pairwise comparisons). This study demonstrates that SNP capture combined with massively parallel sequencing on a benchtop platform can yield sufficient SNP recovery from compromised samples, enabling accurate, extended kinship predictions.

DNA polymerase (dpol) β has served as a model for structural, kinetic, and computational characterization of the DNA synthesis reaction. The laboratory directed by Samuel H. Wilson has utilized a multifunctional approach to analyze the function of this enzyme at the biological, chemical, and molecular levels for nearly 50 years. Over this time, it has become evident that correlating static crystallographic structures of dpol β with solution kinetic measurements is a daunting task. However, aided by computational and spectroscopic approaches, novel and unexpected insights have emerged. While dpols generally insert wrong nucleotides with similar poor efficiencies, their capacity to insert the right nucleotide depends on the identity of the dpol. Accordingly, the ability to choose right from wrong depends on the efficiency of right, rather than wrong, nucleotide insertion. Structures of dpol β in various liganded forms published by the Wilson laboratory, and others, have provided molecular insights into the molecular attributes that hasten correct nucleotide insertion and deter incorrect nucleotide insertion. Computational approaches have bridged the gap between structures of intermediate complexes and provided insights into this basic and essential chemical reaction.

Strecker et al . (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this system has great promise, we note that the well-established biochemistry of Tn7 suggests that the particular system used may insert not only the transposon but also the entire donor plasmid.

Certain retroviruses, including HIV, insert their DNA in a non-random fraction of the host genome via poorly understood selection mechanisms. Here, we develop a biophysical model for retroviral integrations as stochastic and quasi-equilibrium topological reconnections between polymers. We discover that physical effects, such as DNA accessibility and elasticity, play important and universal roles in this process. Our simulations predict that integration is favoured within nucleosomal and flexible DNA, in line with experiments, and that these biases arise due to competing energy barriers associated with DNA deformations. By considering a long chromosomal region in human T-cells during interphase, we discover that at these larger scales integration sites are predominantly determined by chromatin accessibility. Finally, we propose and solve a reaction-diffusion problem that recapitulates the distribution of HIV hot-spots within T-cells. With few generic assumptions, our model can rationalise experimental observations and identifies previously unappreciated physical contributions to retroviral integration site selection., Comment: Accepted in Nat Comm. SI and Movies can be found at https://www2.ph.ed.ac.uk/~dmichiel/

The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.

As the interest in DNA nanotechnology increases, so does the need for larger and more complex DNA structures. In this work we describe two methods of using large, double-stranded (ds) DNA to self-assemble sequence-specific, non-repetitive microscale structures. A model system restructures T7 DNA (40 kb) through sequence-specific biotinylation followed by intramolecular binding to a 40 nm diameter Neutravidin bead to create T7 “rosettes.” This model system informed the creation of “nodal DNA” where “nodes” with single-stranded DNA flaps are attached to a large dsDNA insert so that a complementary oligonucleotide “strap” bridges the two nodes for restructuring to form a DNA “bolo.” In order to do this in high yield, several methodologies were developed, including a protection / deprotection scheme using RNA / RNase H and dialysis chambers that remove excess straps while retaining large DNA molecules. To assess these restructuring processes, the DNA was adsorbed onto supported lipid bilayers, allowing for a visual assay of their structure using single-molecule fluorescence microscopy. Good agreement between the expected and observed fluorescence intensity measurements of the individual features of restructured DNA for both the DNA rosettes and bolos gives us a high degree of confidence that both processes give sequence-specific restructuring of large, dsDNA molecules to create microscale objects.

To understand structural variation for personal genomics, an extensive ensemble of large DNA molecules will be required to span large structural variations. Nanocoding, a whole-genome analysis platform, can analyze large DNA molecules for the construction of physical restriction maps of entire genomes. However, handling of large DNA is difficult and a system is needed to concentrate large DNA molecules, while keeping the molecules intact. Insert technology was developed to protect large DNA molecules during routine cell lysis and molecular biology techniques. However, eluting and concentrating DNA molecules has been difficult in the past. Utilizing 3D printed mesofluidic device, a proof of principle system was developed to elute and concentrate lambda DNA molecules at the interface between a solution and a poly-acrylamide roadblock. The matrix allowed buffer solution to move through the pores in the matrix; however, it slowed down the progression of DNA in the matrix, since the molecules were so large and the pore size was small. Using fluorescence intensity of the insert, 84% of DNA was eluted from the insert and 45% of DNA was recovered in solution from the eluted DNA. DNA recovered was digested with a restriction enzyme to determine that the DNA molecules remained full length during the elution and concentration of DNA.

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains.

DNA mismatch repair (MMR) pathways coordinate the excision and re-synthesis of newly-replicated DNA if a mismatched base-pair has been identified by protein MutS or MutS homologues (MSHs) after replication. DNA excision during MMR is initiated at single-strand breaks (SSBs) in vitro, and several redundant processes have been observed in reconstituted systems which either require a pre-formed SSB in the DNA and/or require a mismatch-activated nicking endonuclease to introduce a new SSB in order to initiate MMR. However, the conditions under which each of these processes may actually occur in living cells have remained obscured by the limitations of current MMR assays. Here we use a novel assay involving chemically-modified oligonucleotide probes to insert targeted DNA ‘mismatches’ directly into the genome of living bacteria to interrogate their replication-coupled repair processes quantitatively in a strand-, orientation-, and mismatched nucleotide-specific manner. This ‘semi-protected oligonucleotide recombination’ (SPORE) assay reveals direct evidence in Escherichia coli of an efficient endonuclease-independent MMR process on the lagging strand—a mechanism that has long-since been considered for lagging-strand repair but never directly shown until now. We find endonuclease-independent MMR is coordinated asymmetrically with respect to the replicating DNA—directed primarily from 3′- of the mismatch—and that repair coordinated from 3′- of the mismatch is in fact the primary mechanism of lagging-strand MMR. While further work is required to explore and identify the molecular requirements for this alternative endonuclease-independent MMR pathway, these findings made possible using the SPORE assay are the first direct report of this long-suspected mechanism in vivo.

In DNA splicing system, the potential effects of sets of restriction enzymes and a ligase that allow DNA molecules to be cleaved and reassociated to produce further molecules are studied. A splicing language depicts the molecules resulting from a splicing system. In this research, a C++ programming code for DNA splicing system with one palindromic restriction enzyme for one and two (non-overlapping) cutting sites is developed. A graphical user interface, GUI is then designed to allow the user to insert the initial DNA string and restriction enzymes to generate the splicing languages which are the result of the computation of the C++ programming. This interface displays the resulting splicing languages, which depict the results from in vitro experiments of the respective splicing system. The results from this research simplify the lenghty manual computation of the resulting splicing languages of DNA splicing systems with one palindromic restriction enzyme.

Activity-based screening of metagenomic DNA libraries is a promising approach to fish out genes encoding novel bioactive compounds/enzymes of industrial importance. The starting point of such functional screening in fosmid vectors is isolation of high molecular weight (HMW) DNA of sufficient purity from diverse environments. Metagenomic DNA isolation protocols mostly employ mechanical cell lysis that yields fragmented DNA. Those established for HMW DNA using enzymatic lysis have not considered samples with high lignocellulose or humic acid content. Enzymes from such environments are in great demand for bioenergy, paper, and related industries. Thus, an improved method was standardized that has three key features, i.e., use of harvested microbial biomass instead of raw samples, removal of humic substances prior to cell lysis by aluminum sulfate flocculation, and enzymatic/chemical lysis of cells with a lysozyme, mutanolysin, proteinase K, and SDS cocktail followed by phenol-chloroform extraction and precipitation of DNA by polyethylene glycol and NaCl. HMW DNA (~ 40 kb) was efficiently isolated from garden and forest soils, rice straw compost, and degrading wood from a hypersaline lake. The humic acid removal efficiency across samples was 96-98%. The isolated DNA was of high quality/purity and could be successfully used in downstream applications like PCR, ligation, and fosmid cloning. In fact, the DNA was directly used without any size selection, for fosmid library preparation with 70-90% efficiency as compared to the control insert. Thus, the method could suitably be used for HMW DNA isolation for the functional screening of enzymes from diverse humic acid-/lignocellulose-rich environments.

Synthetically replicating key biological processes requires the ability to puncture lipid bilayer membranes and to remodel their shape. Recently developed artificial DNA nanopores are one possible synthetic route due to their ease of fabrication. However, an unresolved fundamental question is how DNA nanopores bind to and dynamically interact with lipid bilayers. Here we use single-molecule fluorescence microscopy to establish that DNA nanopores carrying cholesterol anchors insert via a two-step mechanism into membranes. Nanopores are furthermore shown to locally cluster and remodel membranes into nanoscale protrusions. Most strikingly, the DNA pores can function as cytoskeletal components by stabilizing autonomously formed lipid nanotubes. The combination of membrane puncturing and remodeling activity can be attributed to the DNA pores’ tunable transition between two orientations to either span or co-align with the lipid bilayer. This insight is expected to catalyze the development of future functional nanodevices relevant in synthetic biology and nanobiotechnology., DNA nanopores can span lipid bilayers but how they interact with lipids is not known. Here the authors establish at single-molecule level the insertion mechanism and show that DNA nanopores can locally cluster and remodel membranes, and stabilize autonomously formed lipid nanotubes.

Model DNA molecules that contain bulky lesions in both strands have been created, and their properties as substrates of the nucleotide excision repair (NER) system have been analyzed. The modified nucleoside, 5-[3-(4-azido-2,3,5,6-tetrafluorobenzamido)-1-propoxypropyl]-2'-deoxycytidine (dC^(FAB)), or the nonnucleoside fragment, N-[6-(9-anthracenylcarbamoyl)hexanoyl]-3-amino-1,2-propanediol (nAnt), have been inserted as damage in certain positions of the first DNA strand ("0"). The position of N-[6-5(6)-fluoresceinylcarbamoyl]hexanoyl] -3-amino-1,2-propanediol (nFlu) has been varied within the second DNA strand. This residue has been located opposite the removable damaging fragment of the first strand at positions -20, -10, -4, 0, +3, and +8 relative to the first lesion. It has been demonstrated that the presence of nFlu at the -4, 0, or +3 position of the second strand significantly reduces the thermostability of DNA duplexes, especially in the case of nAnt-DNA and completely excludes the possibility of NER-catalyzed excision from dC^(FAB)- and nAnt-containing 137-meric DNA with the second lesion at these positions. The introduction of nFlu at positions -20, -10, or +8 differently affects the excision efficiency of dC^(FAB)- and nAnt-containing fragments from the first strand. The excision efficiency of dC^(FAB)-containing fragments from extended double-damaged DNA is as high as from DNA that contains a single dC^(FAB) damage, while the excision of nAnt-containing fragments occurs with 80-90% lower efficiency from double-damaged DNA occurs from DNA that contains the single nAnt insert. The nFlu insert differently affects the interaction of the sensory XPC-HR23B dimer with dC^(FAB)- and nAnt-containing DNAs, although in all cases, this interaction occurs with increased efficiency compared to that with single-damaged DNAs. No direct correlation between the thermostability of the DNA duplex and XPC-DNA affinity on the one hand, and the excision efficiency of lesions on the other hand has been shown. The absence of the correlation may be caused by both functional features of variable multiprotein complexes involved in the recognition and verification of damage during NER and the sensitivity of the complexes to the structure of the damage and damage-surrounding DNA. The results are important for understanding the NER mechanism of elimination of bulky damage located in both DNA strands.

Base excision DNA repair (BER) is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. BER is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Human endonuclease VIII-like protein 2 (hNEIL2), belonging to the helix–two-turn–helix structural superfamily of DNA glycosylases, is an enzyme uniquely specific for oxidized pyrimidines in non-canonical DNA substrates such as bubbles and loops. The structure of hNEIL2 has not been solved; its closest homologs with known structures are NEIL2 from opossum and from giant mimivirus. Here we analyze the conformational dynamics of free hNEIL2 using a combination of hydrogen/deuterium exchange mass spectrometry, homology modeling and molecular dynamics simulations. We show that a prominent feature of vertebrate NEIL2 – a large insert in its N-terminal domain absent from other DNA glycosylases – is unstructured in solution. It was suggested that helix–two-turn–helix DNA glycosylases undergo open–close transition upon DNA binding, with the large movement of their N- and C-terminal domains, but the open conformation has been elusive to capture. Our data point to the open conformation as favorable for free hNEIL2 in solution. Overall, our results are consistent with the view of hNEIL2 as a conformationally flexible protein, which may be due to its participation in the repair of non-canonical DNA structures and/or to the involvement in functional and regulatory protein–protein interactions.

The Wilms' tumour gene 1 protein (WT1) is a zinc finger transcription factor found indispensable for foetal development. WT1 has also been implicated in the development of tumours in several organ systems, including acute myeloid leukaemia (AML). Four main WT1 isoforms come from 2 alternative splice events. One alternative splice results in the inclusion or exclusion of 3 amino acids, KTS, between zinc fingers 3 and 4 in the WT1 protein. The KTS insert has been extensively investigated due to the functional implications for DNA and RNA binding. In this review, we provide an overview of the research into the isoforms containing or lacking the KTS insert in leukaemic cells, as well as the research into the binding patterns of the WT1 -KTS and +KTS isoforms to DNA and RNA. Finally, we connect the results of the DNA binding research to the ChIP-CHIP and ChIP-Seq investigations into the global genomic binding of the WT1 protein that have recently been performed.

The major difference between viruses and plasmids is the mechanism of transferring their genomic information between host cells. Here, we describe the archaeal plasmid pR1SE from an Antarctic species of haloarchaea that transfers via a mechanism similar to a virus. pR1SE encodes proteins that are found in regularly shaped membrane vesicles, and the vesicles enclose the plasmid DNA. The released vesicles are capable of infecting a plasmid-free strain, which then gains the ability to produce plasmid-containing vesicles. pR1SE can integrate and replicate as part of the host genome, resolve out with fragments of host DNA incorporated or portions of the plasmid left behind, form vesicles and transfer to new hosts. The pR1SE mechanism of transfer of DNA could represent the predecessor of a strategy used by viruses to pass on their genomic DNA and fulfil roles in gene exchange, supporting a strong evolutionary connection between plasmids and viruses. An archaeal plasmid that can be transported in membrane vesicles, similar to a virus, and encodes proteins that can insert into host membranes and membrane vesicles, provides insights into the evolutionary link between plasmids and viruses.