Monomeric DNA Research Articles

Anti-parallel triplexes: Synthesis of 8-aza-7-deazaadenine nucleosides with a 3-aminopropynyl side-chain and its corresponding LNA analog

The phosphoramidites of DNA monomers of 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine (Y) and 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine LNA (Z) are synthesized, and the thermal stability at pH 7.2 and 8.2 of anti-parallel triplexes modified with these two monomers is determined. When, the anti-parallel TFO strand was modified with Y with one or two insertions at the end of the TFO strand, the thermal stability was increased 1.2°C and 3°C at pH 7.2, respectively, whereas one insertion in the middle of the TFO strand decreased the thermal stability 1.4°C compared to the wild type oligonucleotide. In order to be sure that the 3-aminopropyn-1-yl chain was contributing to the stability of the triplex, the nucleobase X without the aminopropynyl group was inserted in the same positions. In all cases the thermal stability was lower than the corresponding oligonucleotides carrying the 3-aminopropyn-1-yl chain, especially at the end of the TFO strand. On the other hand, the thermal stability of the anti-parallel triplex was dramatically decreased when the TFO strand was modified with the LNA monomer analog Z in the middle of the TFO strand (ΔTm=−9.1°C). Also the thermal stability decreased about 6.1°C when the TFO strand was modified with Z and the Watson–Crick strand with adenine-LNA (AL). The molecular modeling results showed that, in case of nucleobases Y and Z a hydrogen bond (1.69 and 1.72Ǻ, respectively) was formed between the protonated 3-aminopropyn-1-yl chain and one of the phosphate groups in Watson–Crick strand. Also, it was shown that the nucleobase Y made a good stacking and binding with the other nucleobases in the TFO and Watson–Crick duplex, respectively. In contrast, the nucleobase Z with LNA moiety was forced to twist out of plane of Watson–Crick base pair which is weakening the stacking interactions with the TFO nucleobases and the binding with the duplex part.

Microwave-assisted phosphitylation of DNA and RNA nucleosides and their analogs.

Microwave-assisted chemical phosphitylation of novel nucleoside analogs containing a ribulose sugar unit was successful with yields ranging from 50% to 79% using 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite as the phosphitylating reagent. The resultant phosphoramidite products remained intact, with no signs of degradation over extended reaction times (up to 60 min) at an elevated temperature (65°C). When the same microwave-mediated phosphitylating protocols were applied to canonical DNA and RNA nucleoside monomers as substrates, using either 2-cyanoethyl-N,N,-diisopropyl chlorophosphoramidite or 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphane with an activator, 40% to 90% yields of DNA and RNA phosphoramidites were obtained within 10 to 15 min. These results demonstrate that microwave-assisted phosphitylation is an efficient alternative to standard phosphitylating conditions that can be expanded and refined to include a variety of substrates.

Streptavidin bound DNA open tube and Zn2+-doped DNA open lattice

DNA is one of the most promising molecules for use in nanotechnology because it has a nanoscale size and also has the ability for self-assembly. In this paper, we discuss the use of free-solution growth for a 1D DNA open tube (OT) and substrate-assisted growth for a 2D open lattice (OL), which can both achieve similar design schemes. We introduced biotinylated OT and OL, which can be bound with streptavidin for visualization, to verify via atomic force microscopy that dimensional structures have in fact been formed. Additionally the coverage ratio controlled by the concentration of the DNA monomer was analyzed to understand the lattice growth on the substrate. The DNA lattices were observed to start growing on the substrate at a concentration of around 1 nM (threshold) and to achieve full coverage at 10 nM (saturation concentration). Finally, the Raman spectra and the current–voltage characteristics of Zn2+-doped OLs were obtained in order to demonstrate the feasibility of using such methods to produce useful materials for nanodevices and biosensors. As [Zn2+] increases above the critical value of 0.5 mM, the Raman peaks gradually decrease. The resistance decreases up to the critical value of [Zn2+], and then decreases [Zn2+] continues to increase.

Locked nucleic acid (LNA) induced effect on the hybridization and fluorescence properties of oligodeoxyribonucleotides modified with nucleobase-functionalized DNA monomers.

LNA and nucleobase-modified DNA monomers are two types of building blocks that are used extensively in oligonucleotide chemistry. However, there are only very few reports in which these two monomer families are used alongside each other. In the present study we set out to characterize the biophysical properties of oligodeoxyribonucleotides in which C5-modified 2'-deoxyuridine or C8-modified 2'-deoxyadenosine monomers are flanked by LNA nucleotides. We hypothesized that the LNA monomers would alter the sugar rings of the modified DNA monomers toward more RNA-like North-type conformations for maximal DNA/RNA affinity and specificity. Indeed, the incorporation of LNA monomers almost invariably results in increased target affinity and specificity relative to the corresponding LNA-free ONs, but the magnitude of the stabilization varies greatly. Introduction of LNA nucleotides as direct neighbors into C5-pyrene-functionalized pyrimidine DNA monomers yields oligonucleotide probes with more desirable photophysical properties as compared to the corresponding LNA-free probes, including more intense fluorescence emission upon target binding and improved discrimination of single nucleotide polymorphisms (SNPs). These hybrid oligonucleotides are therefore promising probes for diagnostic applications.

Bacterial initiators form dynamic filaments on single-stranded DNA monomer by monomer.

DNA replication initiation is mediated across all domains of life by initiator proteins oligomerizing at replication origins. Recently, it was shown that initiators can directly bind single-stranded DNA (ssDNA) and thus might enhance origin melting. In this study, we used single-molecule fluorescence assays to probe the ssDNA binding mechanism of the replication initiator DnaA. Our experiments revealed that DnaA forms a dynamic filament on ssDNA in 3′ to 5′ directionality in the presence of ATP and analogs. After nucleation with a three-monomer seed, monomers dynamically assemble and disassemble one monomer at a time at the 5′ end, each monomer binding three nucleotides of ssDNA. The addition of adjacent double-stranded DnaA binding sites stabilized the DnaA filament on ssDNA. Our results extend the current models of origin melting via DnaA ssDNA interaction.

Abstract 5373: In silico investigation of FOXM1 binding and novel inhibitors in epithelial ovarian cancer (EOC)

Abstract Objective: Using TCGA database, we had demonstrated that aberrantly activated Forkhead box M1 (FOXM1) correlates to worse overall survival in a subgroup of platinum resistant patients. Application of thiostrepton, a natural thiazole antibiotics that inhibits FOXM1 transcription activity, improves the cytotoxic effect of paclitaxel and cisplatinum in ascites cells from platinum-resistant patients. However, the use of thiazole antibiotics in the clinic is hampered by difficulties in synthesis, degradation potential, and solubility issues. In this study, we aim to identify novel FOXM1 small molecule inhibitors which can be a new class of therapeutic agents to address the challenges in treating chemotherapy resistant EOC. Methods: The FOXM1 protein structure was obtained from the Protein Databank (pdb ID: 3GXU) and prepared for docking studies by Autodock Vina 1.0.2. Structural representations of the NCI Diversity Set II in were obtained from the NCI website and converted to a pdb format for screening. Individual pdb files were prepared for docking using the prepare_ligand.py scripts form MGLTools 1.5.4 using the largest non-bonded fragment present. The binding affinity was scored as the free energies of binding in kcal/mol. Three selected compounds (one as negative control) were applied to SKOV3, OVCAR3 and ES2 cells for 72 hrs and sulfarodamine B assay was used to measure the cell viability and FOXM1 expression level was confirmed by RT-PCR and western blot. Results: The FOXM1 structure obtained from 3GXU represented the DNA binding region of FOXM1 and possessed the winged helix fold representative of the Forkhead family of enzymes with two wings in direct contact with DNA. For ease of representation, we described both wings as a dimer and a single wing as a monomer. From this structure, we hypothesized two main models of how thiostrepton binding to FOXM1 could possibly curtail its transcriptional activity. In the first model thiostrepton could bind either of the wings or both wings and prevent association to DNA. In the second model thiostrepton bind the FOXM1: DNA complex and weaken association of FOXM1 to DNA. Subsequently, small molecular inhibitors could also use either of the models to inhibit transcription. To account for both models, the NCI Diversity set was screened against the FOXM1 dimer:DNA complex (39 hits), dimer (11 hits) and monomer (14 hits). Those hits were further classified by chemical structure, biological function and chemical similarities to known molecules that target FOXM1. In cellular cytotoxicity assays, ZINC00990239 showed cytotoxicity with IC50 around 10uM, and downregulation of FOXM1 and transcription of its downstream molecules such as CCNB1. Conclusion: By illustrating the characteristics of an effective small molecule inhibitor of FOXM1 should contain, we have taken the first step towards identification of potential compounds with efficacy similar to thiostrepton. Note: This abstract was not presented at the meeting. Citation Format: Yi Chen, Eliza A. Ruben, Marcia M. Bieber, Jayakumar Rajadas, Nelson N.h Teng. In silico investigation of FOXM1 binding and novel inhibitors in epithelial ovarian cancer (EOC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5373. doi:10.1158/1538-7445.AM2014-5373

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome.

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions.

DNA Looping Provides for “Intersegmental Hopping” by Proteins: A Mechanism for Long-Range Site Localization

Studies on how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites that controls the expression of virulence genes in several human pathogens. Provocatively, we find that Dam travels between its recognition sites most efficiently when those sites are ~500bp apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), which invoke recent reports demonstrating that DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence-specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.

Tetrahedron DNA dendrimers and their encapsulation of gold nanoparticles

DNA dendrimers have achieved increasing attention recently. Previously reported DNA dendrimers used Y-DNA as monomers. Tetrahedron DNA is a rigid tetrahedral cage made of DNA. Herein, we use tetrahedron DNA as monomers to prepare tetrahedron DNA dendrimers. The prepared tetrahedron DNA dendrimers have larger size compared with those made of Y-DNA. In addition, thanks to the central cavity of tetrahedron DNA monomers, some nanoscale structures (e.g., gold nanoparticles) can be encapsulated within tetrahedron DNA monomers. Tetrahedron DNA encapsulated with gold nanoparticles can be further assembled into dendrimers, guiding gold nanoparticles into clusters.

An Efficient Antiviral Strategy for Targeting Hepatitis B Virus Genome Using Transcription Activator-Like Effector Nucleases

The hepatitis B virus (HBV) is a DNA virus that can cause chronic hepatitis B (CHB) in humans. Current therapies for CHB infection are limited in efficacy and do not target the pre-existing viral genomic DNA, which are present in the nucleus as a covalently closed circular DNA (cccDNA) form. The transcription activator-like (TAL) effector nucleases (TALENs) are newly developed enzymes that can cleave sequence-specific DNA targets. Here, TALENs targeting the conserved regions of the viral genomic DNA among different HBV genotypes were constructed. The expression of TALENs in Huh7 cells transfected with monomeric linear full-length HBV DNA significantly reduced the viral production of HBeAg, HBsAg, HBcAg, and pgRNA, resulted in a decreased cccDNA level and misrepaired cccDNAs without apparent cytotoxic effects. The anti-HBV effect of TALENs was further demonstrated in a hydrodynamic injection-based mouse model. In addition, an enhanced antiviral effect with combinations of TALENs and interferon-α (IFN-α) treatment was observed and expression of TALENs restored HBV suppressed IFN-stimulated response element-directed transcription. Taken together, these data indicate that TALENs can specifically target and successfully inactivate the HBV genome and are potently synergistic with IFN-α, thus providing a potential therapeutic strategy for treating CHB infection.

Programmable Imaging Amplification via Nanoparticle‐Initiated DNA Polymerization

DNA growth on nanoparticles: Nanoparticle-mediated molecular recognition and DNA polymerization are integrated for extracellular matrix (ECM) imaging via programmable signal amplification. By using DNA monomers labeled with identical or different imaging reagents, it is promising to achieve single- or multiple-modality imaging of the ECM.

Abstract 577: DNA replication origin of human alphoid centromere regions .

Abstract The human centromere DNA consist of 171 bp AT-rich-alpha satellite DNA that tandemly arranged into higher order repeat units that is diverged at each centromere, extend from 200kb to 4MB. As the chromosomal site that regulates accurate chromosome segregation, maintaining the correct and coordinated DNA replication prior to cell division is critical. This process should ensure that whole genetic information transferred to daughter cell faithfully. Irregularities on replication process are leading to the mitosis defect and apoptosis. Flanking regions of alpha-satellite DNA arrays are organized in heterochromatin domains that are co-localized with other types of centromeric repeats, i.e. satellite II and gamma-satellite DNA. Previous studies indicate that centromeric DNA replicates in the middle to late S-phase. However little is known what determines activities and positions of replicators on chromatin domains. In this work, we addressed the mechanism of replication on the centromere and other repeat sequences on pericentromeric regions using two different approaches: i) origin mapping using nascent-strand DNA and ii) DNA-protein interaction using chromatin-immunoprecipitation (ChIP) assay. Our results detect active origin of replication within alpha-satellite DNA monomers of centromere, as well as on gamma-satellite repeats. In contrast, satellite II repeats representing centromere-adjacent heterochromatin do not exhibit a significant activity as origin of replication. The accumulation of modified-Histone proteins indicated dynamic activity of DNA arrays. Moreover, DNA replication proteins are formed complex with centromere proteins, presented direct evident on DNA replication activity on centromere. In summary, data indicated position of replicator within alphoid centromere and other large tandem repeat arrays on pericentromeric region with condense chromatin structure, thus suggested role of epigenetic modifications on DNA arrays propagation. Citation Format: Indri Erliandri, Haiqing Fu, Jung-Hyun Kim, Mirit Aladjem, William Earnshaw, Ramaiah Nagaraja, David Schlessinger, Vladimir Larionov. DNA replication origin of human alphoid centromere regions . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 577. doi:10.1158/1538-7445.AM2013-577

RNAiFOLD: A CONSTRAINT PROGRAMMING ALGORITHM FOR RNA INVERSE FOLDING AND MOLECULAR DESIGN

Synthetic biology is a rapidly emerging discipline with long-term ramifications that range from single-molecule detection within cells to the creation of synthetic genomes and novel life forms. Truly phenomenal results have been obtained by pioneering groups--for instance, the combinatorial synthesis of genetic networks, genome synthesis using BioBricks, and hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment, biomolecular self-assembly pathways, etc. Such work strongly suggests that nanotechnology and synthetic biology together seem poised to constitute the most transformative development of the 21st century. In this paper, we present a Constraint Programming (CP) approach to solve the RNA inverse folding problem. Given a target RNA secondary structure, we determine an RNA sequence which folds into the target structure; i.e. whose minimum free energy structure is the target structure. Our approach represents a step forward in RNA design--we produce the first complete RNA inverse folding approach which allows for the specification of a wide range of design constraints. We also introduce a Large Neighborhood Search approach which allows us to tackle larger instances at the cost of losing completeness, while retaining the advantages of meeting design constraints (motif, GC-content, etc.). Results demonstrate that our software, RNAiFold, performs as well or better than all state-of-the-art approaches; nevertheless, our approach is unique in terms of completeness, flexibility, and the support of various design constraints. The algorithms presented in this paper are publicly available via the interactive webserver http://bioinformatics.bc.edu/clotelab/RNAiFold; additionally, the source code can be downloaded from that site.

The roles of the monomer length and nucleotide context of plant tandem repeats in nucleosome positioning

Similar to regularly spaced nucleosomes in chromatin, long tandem DNA arrays are composed of regularly alternating monomers that have almost identical primary DNA structures. Such a similarity in the structural organization makes these arrays especially interesting for studying the role of intrinsic DNA preferences in nucleosome positioning. We have studied the nucleosome formation potential of DNA tandem repeat families with different monomer lengths (ML). In total, 165 plant tandem repeat families from the PlantSat database (http://w3lamc.umbr.cas.cz/PlantSat/) were divided into two classes based on the number of nucleosome repeats in one DNA monomer. For predicting nucleosome formation potential, we developed the Phase method, which combines the advantages of multiple bioinformatics models. The Phase method was able to distinguish interfamily differences and intrafamily monomer variation and identify the influence of nucleotide context on nucleosome formation potential. Three main types of nucleosome arrangement in DNA tandem repeat arrays – regular, partially regular (partial), and flexible – were distinguished among a great variety of Phase profiles. The regular type, in which all nucleosomes of the monomer array are positioned in a context-dependent manner, is the most representative type of the class 1 families, with ML equal to or a multiple of the nucleosome repeat length (NRL). In the partially regular type, nucleotide context influences the positioning of only a subset of nucleosomes. The influence of the nucleotide context on nucleosome positioning has the least effect in the flexible type, which contains the greatest number of families (65). The majority of these families belong to class 2 and have nonmultiple ML to NRL ratios.

High‐Yield Synthesis of Silver Nanoclusters Protected by DNA Monomers and DFT Prediction of their Photoluminescence Properties

Basic evidence is provided for the benefits of using cytosine-rich DNA strands as scaffolds for fluorescent silver nanoclusters. The DFT-calculated fluorescence spectra of silver nanoclusters (AgNCs) protected by DNA monomers (dC, dA, dT, and dG) were in good agreement with the experimentally obtained spectra (see picture), showing that only the use of cytosine-rich DNA strands as scaffolds gives fluorescent nanoclusters.

High‐Yield Synthesis of Silver Nanoclusters Protected by DNA Monomers and DFT Prediction of their Photoluminescence Properties

Ein klarer Beweis für die Vorteile der Verwendung Cytosin-reicher DNA-Stränge zur Stabilisierung fluoreszierender Ag-Nanocluster wurde erbracht. Die berechneten Fluoreszenzspektren von mit DNA-Monomeren (dC, dA, dT und dG) geschützten Silber-Nanoclustern (AgNCs) stimmten gut mit den experimentellen Spektren überein (siehe Bild) und zeigen, dass nur die Verwendung Cytosin-reicher DNA-Stränge zu fluoreszierenden NCs führt.

Hybridization chain reaction-based instantaneous derivatization technology for chemiluminescence detection of specific DNA sequences

We propose here a new amplifying strategy that uses hybridization chain reaction (HCR) to detect specific sequences of DNA, where stable DNA monomers assemble on the magnetic beads only upon exposure to a target DNA. Briefly, in the HCR process, two complementary stable species of hairpins coexist in solution until the introduction of initiator reporter strands triggers a cascade of hybridization events that yield nicked double helices analogous to alternating copolymers. Moreover, a "sandwich-type" detection strategy is employed in our design. Magnetic beads, which are functionalized with capture DNA, are reacted with the target, and sandwiched with the above nicked double helices. Then, chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), and the guanine nucleotides within the target DNA, reporter strands and DNA monomers for the generation of light. Our results clearly show that the amplification detection of specific sequences of DNA achieves a better performance (e.g. wide linear response range, low detection limit, and high specificity) as compared to the traditional sandwich type (capture/target/reporter) assays. Upon modification, the approach presented could be extended to detect other types of targets. We believe that this simple technique is promising for improving medical diagnosis and treatment.

Systematic Studies of UV Stability and Photopolymerization Efficiency of DNA‐Based Nanomaterials

DNA has gained acceptance as an attractive nanomaterial building block due to its versatility and manipulability in design and synthesis. Our group has synthesized a variety of branched DNA monomers including X-shaped DNA (X-DNA) and Y-shaped DNA (Y-DNA), which have well-defined nanoscale geometries. 7] Various functional moieties can also be conjugated onto the DNA monomers, allowing them to have diverse applications. In particular, a photoreactive group can be covalently attached to each arm to allow for UV-initiated polymerization (photopolymerization). Photopolymerization provides a rapid, one-step route for polymerization at room temperature in an aqueous environment, thus it can be easily controlled spatially and temporally. 9] Using photopolymerization, we have successfully constructed DNA nanospheres from XDNA monomers and multifunctional DNA polymers from anisotropic, branched, and crosslinkable (ABC) DNA monomers. Despite the advantages of photopolymerization, reactive oxygen species (ROS) created during the photopolymerization process, including singlet oxygen (O2) and hydroxyl radicals (·OH), cause DNA damage, which may compromise the structural integrity of the photopolymerized DNA nanomaterials. Photoinitiators, commonly used to initiate photopolymerization, are more prone to radical formation, and these free radicals create more ROS that may further cause DNA damage. The mechanisms of DNA damage by ROS include cleavage of sugar-phosphate backbone, disruption of hydrogen bonds by oxidative base modification, cleavage of the nitrogenous base from the 2’ carbon in the deoxyribose sugar, and cleavage of the phosphodiester bonds in the DNA backbone. These disrupted bonds compromise the DNA material integrity. To address these issues, several photoreaction parameters have been studied in terms of their effects on DNA damage; however, most of these studies have only focused on plasmid DNA. For example, Anseth and co-workers reported that wavelength of UV affected DNA damage: short wavelengths (254 nm) turned some intact plasmids into the open-circle nicked form, but long wavelengths (365 nm) had minimal impact on DNA integrity. Park and colleagues revealed that photoreaction time also affected DNA damage: with longer irradiation time, plasmid DNA suffered more damage. Many other studies have investigated the relationshiop between UV light and DNA exclusively on the genetic and cellular level. To the best of our knowledge, this report is the first systematic study of UV damage on DNA building blocks (monomers), rather than plasmids, during photopolymerization. To establish optimal conditions for our DNA photopolymerization system, we first quantified damage on DNA monomers during photopolymerization and second studied the polymerization efficiency of photopolymerizable DNA monomers. More specifically, we focused on parameters relating to: 1) the DNA buildling blocks and 2) the photoreaction conditions. With respect to the DNA building blocks, we varied DNA concentration, structure, and condition of a protectant [i.e. poly(ethylene glycol)] ; for the photoreaction conditions, we examined UV reaction time, photoinitiator concentration, and UV intensity (Scheme 1).

Rescue of a Porcine Anellovirus (Torque Teno Sus Virus 2) from Cloned Genomic DNA in Pigs

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.

Complementation between inactive fragments of SssI DNA methyltransferase

BackgroundSilencing mammalian genes by targeted DNA (cytosine-5) methylation of selected CG sites in the genome would be a powerful technique to analyze epigenomic information and to study the roles of DNA methylation in physiological and pathological states. A promising approach of targeted DNA methylation is based on the ability of split fragments of a monomeric DNA methyltransferase (C5-MTase) to associate and form active enzyme. A few C5-MTases of different specificities have been shown to possess the ability of fragment complementation, but a demonstration of this phenomenon for a C5-MTase, which has CG specificity and thus can be targeted to methylate any CG site, has been lacking. The purpose of this study was to test whether the CG-specific prokaryotic C5-MTase M.SssI shows the phenomenon of fragment complementation.ResultsWe show that truncated inactive N-terminal fragments of M.SssI can assemble with truncated inactive C-terminal fragments to form active enzyme in vivo when produced in the same E. coli cell. Overlapping and non-overlapping fragments as well as fragments containing short appended foreign sequences had complementation capacity. In optimal combinations C-terminal fragments started between conserved motif VIII and the predicted target recognizing domain of M.SssI. DNA methyltransferase activity in crude extracts of cells with the best complementing fragment pairs was ~ 4 per cent of the activity of cells producing the full length enzyme. Fusions of two N-terminal and two C-terminal fragments to 21.6 kDa zinc finger domains only slightly reduced complementation ability of the fragments.ConclusionsThe CG-specific DNA methyltransferase M.SssI shows the phenomenon of fragment complementation in vivo in E. coli. Fusion of the split fragments to six unit zinc finger domains does not substantially interfere with the formation of active enzyme. These observations and the large number of complementing fragment combinations representing a wide range of MTase activity offer the possibility to develop M.SssI into a programmable DNA methyltransferase of high specificity.