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    Understanding intercalative modulation of G-rich sequence folding: solution structure of a TINA-conjugated antiparallel DNA triplex.
    (Oxford University Press, 2024-01-28) Garavís M; Edwards PJB; Serrano-Chacón I; Doluca O; Filichev VV; González C
    We present here the high-resolution structure of an antiparallel DNA triplex in which a monomer of para-twisted intercalating nucleic acid (para-TINA: (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) is covalently inserted as a bulge in the third strand of the triplex. TINA is a potent modulator of the hybridization properties of DNA sequences with extremely useful properties when conjugated in G-rich oligonucleotides. The insertion of para-TINA between two guanines of the triplex imparts a high thermal stabilization (ΔTM = 9ºC) to the structure and enhances the quality of NMR spectra by increasing the chemical shift dispersion of proton signals near the TINA location. The structural determination reveals that TINA intercalates between two consecutive triads, causing only local distortions in the structure. The two aromatic moieties of TINA are nearly coplanar, with the phenyl ring intercalating between the flanking guanine bases in the sequence, and the pyrene moiety situated between the Watson-Crick base pair of the two first strands. The precise position of TINA within the triplex structure reveals key TINA-DNA interactions, which explains the high stabilization observed and will aid in the design of new and more efficient binders to DNA.
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    DeepCAC: a deep learning approach on DNA transcription factors classification based on multi-head self-attention and concatenate convolutional neural network
    (BioMed Central Ltd, 2023-09-18) Zhang J; Liu B; Wu J; Wang Z; Li J
    Understanding gene expression processes necessitates the accurate classification and identification of transcription factors, which is supported by high-throughput sequencing technologies. However, these techniques suffer from inherent limitations such as time consumption and high costs. To address these challenges, the field of bioinformatics has increasingly turned to deep learning technologies for analyzing gene sequences. Nevertheless, the pursuit of improved experimental results has led to the inclusion of numerous complex analysis function modules, resulting in models with a growing number of parameters. To overcome these limitations, it is proposed a novel approach for analyzing DNA transcription factor sequences, which is named as DeepCAC. This method leverages deep convolutional neural networks with a multi-head self-attention mechanism. By employing convolutional neural networks, it can effectively capture local hidden features in the sequences. Simultaneously, the multi-head self-attention mechanism enhances the identification of hidden features with long-distant dependencies. This approach reduces the overall number of parameters in the model while harnessing the computational power of sequence data from multi-head self-attention. Through training with labeled data, experiments demonstrate that this approach significantly improves performance while requiring fewer parameters compared to existing methods. Additionally, the effectiveness of our approach  is validated in accurately predicting DNA transcription factor sequences.
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    Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.
    (Springer Nature Limited, 2023-10-11) Harjes S; Kurup HM; Rieffer AE; Bayarjargal M; Filitcheva J; Su Y; Hale TK; Filichev VV; Harjes E; Harris RS; Jameson GB
    The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.
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    Sarcoid within the oral cavity of a horse.
    (Elsevier B.V., 2024-02-01) Munday JS; Lewis MC; Leyland MH
    Equine sarcoids are common skin tumors that are thought to be caused by cross-species infection by bovine papillomaviruses (BPV). A 16-year-old horse developed a 1cm diameter mandibular gingival mass opposite the right second premolar tooth (406) and a 2cm diameter mass close to the commissure of the lips on the same side of the mouth. The right cheek was diffusely thickened. Histology of the smaller mass revealed a proliferation of mesenchymal cells covered by hyperplastic epithelium that formed thick rete pegs. BPV2 DNA was amplified from the mass. Although the mass had been incompletely excised, there was no recurrence after 5 months. The histological features and detection of BPV2 DNA is consistent with a diagnosis of equine sarcoid. Sarcoids have not previously been reported in the oral cavity of horses. It is hypothesized that trauma to the mouth may have been important for sarcoid development. Additionally, different BPV types may have variable ability to infect the gingiva. While rare, sarcoids are a differential for an oral mass in a horse.
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    Molecular typing of Leptospira spp. in farmed and wild mammals reveals new host-serovar associations in New Zealand.
    (Taylor and Francis Group, 2024-01-01) Wilkinson DA; Edwards M; Shum C; Moinet M; Anderson NE; Benschop J; Nisa S
    AIMS: To apply molecular typing to DNA isolated from historical samples to determine Leptospira spp. infecting farmed and wild mammals in New Zealand. MATERIALS AND METHODS: DNA samples used in this study were extracted from urine, serum or kidney samples (or Leptospira spp. cultures isolated from them) collected between 2007 and 2017 from a range of domestic and wildlife mammalian species as part of different research projects at Massey University. Samples were included in the study if they met one of three criteria: samples that tested positive with a lipL32 PCR for pathogenic Leptospira; samples that tested negative by lipL32 PCR but were recorded as positive to PCR for pathogenic Leptospira in the previous studies; or samples that were PCR-negative in all studies but were from animals with positive agglutination titres against serogroup Tarassovi. DNA samples were typed using PCR that targeted either the glmU or gyrB genetic loci. The resulting amplicons were sequenced and typed relative to reference sequences. RESULTS: We identified several associations between mammalian hosts and Leptospira strains/serovars that had not been previously reported in New Zealand. Leptospira borgpetersenii strain Pacifica was found in farmed red deer (Cervus elaphus) samples, L. borgpetersenii serovars Balcanica and Ballum were found in wild red deer samples, Leptospira interrogans serovar Copenhageni was found in stoats (Mustela erminea) and brushtail possums (Trichosurus vulpecula), and L. borgpetersenii was found in a ferret (Mustela putorius furo). Furthermore, we reconfirmed previously described associations including dairy cattle with L. interrogans serovars Copenhageni and Pomona and L. borgpetersenii serovars Ballum, Hardjo type bovis and strain Pacifica, sheep with L. interrogans serovar Pomona and L. borgpetersenii serovar Hardjo type bovis, brushtail possum with L. borgpetersenii serovar Balcanica, farmed deer with L. borgpetersenii serovar Hardjo type bovis and hedgehogs (Erinaceus europaeus) with L. borgpetersenii serovar Ballum. CONCLUSIONS: This study provides an updated summary of host-Leptospira associations in New Zealand and highlights the importance of molecular typing. Furthermore, strain Pacifica, which was first identified as Tarassovi using serological methods in dairy cattle in 2016, has circulated in animal communities since at least 2007 but remained undetected as serology is unable to distinguish the different genotypes. CLINICAL RELEVANCE: To date, leptospirosis in New Zealand has been diagnosed with serological typing, which is deficient in typing all strains in circulation. Molecular methods are necessary to accurately type strains of Leptospira spp. infecting mammals in New Zealand.
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    Optical microlever assisted DNA stretching
    (Optica Publishing Group, 2021-08-02) Andrew P-K; Raudsepp A; Fan D; Staufer U; Williams MAK; Avci E
    Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.
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    Growth condition-dependent differences in methylation imply transiently differentiated DNA methylation states in Escherichia coli
    (Oxford University Press on behalf of the Genetics Society of America, 2023-02) Breckell GL; Silander OK
    DNA methylation in bacteria frequently serves as a simple immune system, allowing recognition of DNA from foreign sources, such as phages or selfish genetic elements. However, DNA methylation also affects other cell phenotypes in a heritable manner (i.e. epigenetically). While there are several examples of methylation affecting transcription in an epigenetic manner in highly localized contexts, it is not well-established how frequently methylation serves a more general epigenetic function over larger genomic scales. To address this question, here we use Oxford Nanopore sequencing to profile DNA modification marks in three natural isolates of Escherichia coli. We first identify the DNA sequence motifs targeted by the methyltransferases in each strain. We then quantify the frequency of methylation at each of these motifs across the entire genome in different growth conditions. We find that motifs in specific regions of the genome consistently exhibit high or low levels of methylation. Furthermore, we show that there are replicable and consistent differences in methylated regions across different growth conditions. This suggests that during growth, E. coli transiently differentiate into distinct methylation states that depend on the growth state, raising the possibility that measuring DNA methylation alone can be used to infer bacterial growth states without additional information such as transcriptome or proteome data. These results show the utility of using Oxford Nanopore sequencing as an economic means to infer DNA methylation status. They also provide new insights into the dynamics of methylation during bacterial growth and provide evidence of differentiated cell states, a transient analog to what is observed in the differentiation of cell types in multicellular organisms.
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    DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies
    (Beilstein-Institut, 2021-03-29) Su Y; Bayarjargal M; Hale TK; Filichev VV; Kumar P; Brown T
    Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3',5'-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.
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    Studies towards thermodynamically stable G-quadruplexes embedded in canonical DNA duplexes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Chilton, Bruce
    DNA is the polymer responsible for the storage of genetic information and, ultimately, all processes that occur within the cell. Our understanding of DNA structure and function has developed considerably, but some areas are still unclear. In particular, a range of non-canonical DNA secondary structures such as G-quadruplexes (G4s), i-motifs and triplexes, have also been shown to form in genomic DNA sequences and these structures also appear to have a role in genome function. Better understanding of the interactions of non-canonical secondary structures is hindered by their transient nature in the context of larger DNA structures, making it difficult to accurately study them using in vitro analytical techniques (e.g., NMR spectroscopy, X-ray crystallography, etc.). They are typically less thermodynamically stable than canonical DNA duplexes and are formed within the genome only in equilibrium with many secondary structures, typically favouring the canonical duplex. They can often only be formed reliably under specific conditions in vitro (e.g., single-stranded, low pH etc.). This thesis presents several strategies designed to stabilise non-canonical G4 secondary structures, which are of interest because they are often found in the promoter regions of oncogenes. The most commonly used existing G4 stabilisation technique utilises small-molecule ligands which specifically bind to and stabilise G4 structures. This thesis includes an investigation into both a widely used G4-binding ligand and several newly developed ligands, but their potential to block binding sites and disrupt G4 topology makes them less suitable for our intended applications. Hydrophobic modifications can encourage aggregation of DNA strands and therefore increase stability of secondary structures. Hydrophobic phosphate modifications in G4s proved effective at disrupting duplexes and stabilising G4s but was limited by coupling efficiency of the modification and resulting difficulties with purification. Intentional mismatches in the G4-forming sequence were introduced by inverting sequence direction or incorporating α-anomers of nucleotides. This strategy was able to completely disrupt duplex formation while preserving G4 structures, but modification sites have to be carefully considered to avoid significant changes in G4 topology. Internal cross-links were incorporated into DNA using modified nucleotides designed for copper(I)-catalysed azide-alkyne cycloaddition. These cross-links prevent G4 structures from unfolding, but the location of these cross-links must also be carefully considered to prevent disruption of the native G4 topology and blocking protein binding sites. All three of these methods present potential routes for stabilising G4s within larger DNA structures. Furthermore, all three modifications could potentially be expanded to stabilise other non-canonical structures, such as imotifs or triplexes.
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    Characterisation of epigenomic variation in natural isolates of E. coli : a thesis submitted in partial fulfilment of the requirements for the degree of Ph.D in Genetics, Massey University, College of Science, School of Natural Sciences, Auckland
    (Massey University, 2023) Breckell, Georgia
    DNA methylation is ubiquitous in bacteria and has a range of roles including self versus non-self recognition, DNA repair, and regulation of gene expression in response to internal and external cues. Regulation of gene expression by DNA methylation can lead to the establishment of phenotypic variation in otherwise isogenic populations. Until recently methods for the genome-wide study of DNA methylation in bacteria have been limited and therefore the full extent of DNA methylation's role in bacterial genomes is not well understood. In this thesis I use Oxford Nanopore Technologies sequencing to investigate the presence and activity of DNA methyltransferase in natural isolates of E. coli. The first aim of this thesis is to produce high quality genome assemblies that can be used to determine methylation patterns. To achieve this, in Chapter 2 I first use in silico methods to quantify the effects of different read length characteristics on assembly quality. I then optimise DNA isolation and library prep methods to obtain high quality DNA. In Chapter 3 I apply the results of Chapter 2 to sequence 49 natural isolates of E. coli from across the E. coli clade. I next benchmark five genome assembly methods for assembly accuracy. I base accuracy on five metrics designed to measure both the overall structural accuracy and the sequence accuracy of each assembly. The large number of isolates (49) used in this study, allows identification of the strengths associated with each assembly method. These results quantitatively describe best practices for bacterial genome assembly and highlight the current variability in genome assembly accuracy and therefore the importance of tailoring assembly methods to the study objectives. Finally, in chapter 4 I use the data produced in Chapter 3 to investigate DNA methylation in three E. coli natural isolates. After in silico identification of all the methyltransferases in each genome, I show that the activity of all predicted methyltransferases can be detected, as well as the activity of unexpected putative methyltransferases which are present in our isolates. Finally, I show that the genome wide DNA methylation patterns show consistent differences across growth conditions. These results suggest that E. coli exhibits transient DNA methylation patterns depending on growth environment and state. Overall this thesis establishes methods for assessing genome assemblies and broadens our understanding of genome wide DNA methylation patterns and the dynamics of these patterns in E. coli. Additionally this work provides insight into the possibility of transient epigenetic differentiation in E. coli which is reflected in the DNA methylation patterns across the genome.