Real-time audio and visual display of the Coronavirus genome.

BACKGROUND: This paper describes a web based tool that uses a combination of sonification and an animated display to inquire into the SARS-CoV-2 genome. The audio data is generated in real time from a variety of RNA motifs that are known to be important in the functioning of RNA. Additionally, metadata relating to RNA translation and transcription has been used to shape the auditory and visual displays. Together these tools provide a unique approach to further understand the metabolism of the viral RNA genome. This audio provides a further means to represent the function of the RNA in addition to traditional written and visual approaches. RESULTS: Sonification of the SARS-CoV-2 genomic RNA sequence results in a complex auditory stream composed of up to 12 individual audio tracks. Each auditory motive is derived from the actual RNA sequence or from metadata. This approach has been used to represent transcription or translation of the viral RNA genome. The display highlights the real-time interaction of functional RNA elements. The sonification of codons derived from all three reading frames of the viral RNA sequence in combination with sonified metadata provide the framework for this display. Functional RNA motifs such as transcription regulatory sequences and stem loop regions have also been sonified. Using the tool, audio can be generated in real-time from either genomic or sub-genomic representations of the RNA. Given the large size of the viral genome, a collection of interactive buttons has been provided to navigate to regions of interest, such as cleavage regions in the polyprotein, untranslated regions or each gene. These tools are available through an internet browser and the user can interact with the data display in real time. CONCLUSION: The auditory display in combination with real-time animation of the process of translation and transcription provide a unique insight into the large body of evidence describing the metabolism of the RNA genome. Furthermore, the tool has been used as an algorithmic based audio generator. These audio tracks can be listened to by the general community without reference to the visual display to encourage further inquiry into the science.

The interactions of novel mononuclear platinum-based complexes with DNA.

BACKGROUND: Cisplatin has been widely used for the treatment of cancer and its antitumour activity is attributed to its capacity to form DNA adducts, predominantly at guanine residues, which impede cellular processes such as DNA replication and transcription. However, there are associated toxicity and drug resistance issues which plague its use. This has prompted the development and screening of a range of chemotherapeutic drug analogues towards improved efficacy. The biological properties of three novel platinum-based compounds consisting of varying cis-configured ligand groups, as well as a commercially supplied compound, were characterised in this study to determine their potential as anticancer agents. METHODS: The linear amplification reaction was employed, in conjunction with capillary electrophoresis, to quantify the sequence specificity of DNA adducts induced by these compounds using a DNA template containing telomeric repeat sequences. Additionally, the DNA interstrand cross-linking and unwinding efficiency of these compounds were assessed through the application of denaturing and native agarose gel electrophoresis techniques, respectively. Their cytotoxicity was determined in HeLa cells using a colorimetric cell viability assay. RESULTS: All three novel platinum-based compounds were found to induce DNA adduct formation at the tandem telomeric repeat sequences. The sequence specificity profile at these sites was characterised and these were distinct from that of cisplatin. Two of these compounds with the enantiomeric 1,2-diaminocyclopentane ligand (SS and RR-DACP) were found to induce a greater degree of DNA unwinding than cisplatin, but exhibited marginally lower DNA cross-linking efficiencies. Furthermore, the RR-isomer was more cytotoxic in HeLa cells than cisplatin. CONCLUSIONS: The biological characteristics of these compounds were assessed relative to cisplatin, and a variation in the sequence specificity and a greater capacity to induce DNA unwinding was observed. These compounds warrant further investigations towards developing more efficient chemotherapeutic drugs.

A website to identify shared genes in Saccharomyces cerevisiae homozygous deletion library screens.

BACKGROUND: The homozygous yeast deletion library includes approximately 4800 diploid strains each containing one deleted non-essential gene. Hundreds of publications have arisen through experimentation using this genome-wide biological resource. As part of this work over 677 genesets have been collated from these experiments representing the phenotypic responses of the library to a diverse set of chemical and physical challenges. DESCRIPTION: A website called the Saccharomyces cerevisiae Homozygous Deletion Library Tools (ScHo DeLiTo-96) has been developed with the primary goal of browsing and identifying genes shared between these responsive phenotypes (available at yeastdb.org ). Geneset comparisons have been performed for each phenotype against all others to identify common genes. Genesets and other curated information are stored in a relational database and a website interface allows users to query and browse the data in an intuitive way to reveal commonality between selected phenotypic responses. The most commonly occurring genes in all of the stored phenotypes are highly over-represented in the GO slim term "cellular ion homeostasis" indicating that genes shared between phenotypes may highlight a common cellular response. Additionally, user derived genesets can be uploaded and intersected against the stored data to reveal common responses which may otherwise have been obscure. CONCLUSION: These tools provide a simple method to perform niche enquiries between datasets derived from the yeast deletion library.

An auditory display tool for DNA sequence analysis.

BACKGROUND: DNA Sonification refers to the use of an auditory display to convey the information content of DNA sequence data. Six sonification algorithms are presented that each produce an auditory display. These algorithms are logically designed from the simple through to the more complex. Three of these parse individual nucleotides, nucleotide pairs or codons into musical notes to give rise to 4, 16 or 64 notes, respectively. Codons may also be parsed degenerately into 20 notes with respect to the genetic code. Lastly nucleotide pairs can be parsed as two separate frames or codons can be parsed as three reading frames giving rise to multiple streams of audio. RESULTS: The most informative sonification algorithm reads the DNA sequence as codons in three reading frames to produce three concurrent streams of audio in an auditory display. This approach is advantageous since start and stop codons in either frame have a direct affect to start or stop the audio in that frame, leaving the other frames unaffected. Using these methods, DNA sequences such as open reading frames or repetitive DNA sequences can be distinguished from one another. These sonification tools are available through a webpage interface in which an input DNA sequence can be processed in real time to produce an auditory display playable directly within the browser. The potential of this approach as an analytical tool is discussed with reference to auditory displays derived from test sequences including simple nucleotide sequences, repetitive DNA sequences and coding or non-coding genes. CONCLUSION: This study presents a proof-of-concept that some properties of a DNA sequence can be identified through sonification alone and argues for their inclusion within the toolkit of DNA sequence browsers as an adjunct to existing visual and analytical tools.

Characterisation of the DNA sequence specificity, cellular toxicity and cross-linking properties of novel bispyridine-based dinuclear platinum complexes.

BACKGROUND: The anti-tumour activity of cisplatin is thought to be a result of its capacity to form DNA adducts which prevent cellular processes such as DNA replication and transcription. These DNA adducts can effectively induce cancer cell death, however, there are a range of clinical side effects and drug resistance issues associated with its use. In this study, the biological properties of three novel dinuclear platinum-based compounds (that contain alkane bridging linkers of eight, ten and twelve carbon atoms in length) were characterised to assess their potential as anticancer agents. METHODS: The properties of these compounds were determined using a DNA template containing seven tandem telomeric repeat sequences. A linear amplification reaction was used in combination with capillary electrophoresis to quantify the sequence specificity of DNA adducts formed by these compounds at base pair resolution. The DNA cross-linking ability of these compounds was assessed using denaturing agarose gel electrophoresis and cytotoxicity was determined in HeLa cells using a colorimetric cell viability assay. RESULTS: The dinuclear compounds were found to preferentially form DNA adducts at guanine bases and they exhibited different damage intensity profiles at the telomeric repeat sequences compared to that of cisplatin. The dinuclear compounds were found to exhibit a low level of cytotoxicity relative to cisplatin and their cytotoxicity increased as the linker length increased. Conversely, the interstrand cross-linking efficiency of the dinuclear compounds increased as the linker length decreased and the compound with the shortest alkane linker was six-fold more effective than cisplatin. CONCLUSIONS: Since the bifunctional compounds exhibit variation in sequence specificity of adduct formation and a greater ability to cross-link DNA relative to cisplatin they warrant further investigation towards the goal of developing new cancer chemotherapeutic agents.

Twenty years later, the evolution of origami DNA.

Twenty years ago Trends in Biochemical Sciences published an innovative origami template for modeling the DNA double helix. Presented here are adaptations of this, including a chemical structure designed for transparent acetate, plus hybrid DNA/RNA, RNA hairpin, and single-stranded models. These models are useful teaching tools for understanding the 3D structure of DNA.

Oxidant-induced cell-cycle delay in Saccharomyces cerevisiae: the involvement of the SWI6 transcription factor.

Cells treated with low doses of linoleic acid hydroperoxide (LoaOOH) exhibit a cell-cycle delay that may provide a mechanism to overcome oxidative stress. Strains sensitive to LoaOOH from the genome-wide deletion collection were screened to identify deletants in which the cell-cycle delay phenotype was reduced. Forty-seven deletants were identified that were unable to mount the normal delay response, implicating the product of the deleted gene in the oxidant-mediated cell-cycle delay of the wild-type. Of these genes, SWI6 was of particular interest due to its role in cell-cycle progression through Start. The swi6 deletant strain was delayed on entry into the cell cycle in the absence of an oxidant, and oxidant addition caused no further delay. Transforming the swi6 deletant with SWI6 on a plasmid restored the G1 arrest in response to LoaOOH, indicating that Swi6p is involved in oxidant sensing leading to cell division delay. Micro-array studies identified genes whose expression in response to LoaOOH depended on SWI6. The screening identified 77 genes that were upregulated in the wild-type strain and concurrently downregulated in the swi6 deletant treated with LoaOOH. These data show that functions such as heat shock response, and glucose transport are involved in the response.

Involvement of oxidative stress response genes in redox homeostasis, the level of reactive oxygen species, and ageing in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants lacking oxidative stress response genes were used to investigate which genes are required under normal aerobic conditions to maintain cellular redox homeostasis, using intracellular glutathione redox potential (glutathione E(h)) to indicate the redox environment of the cells. Levels of reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) were also assessed by FACS using dihydroethidium and rhodamine 123 as fluorescent probes. Cells became more oxidised as strains shifted from exponential growth to stationary phase. During both phases the presence of reduced thioredoxin and the activity of glutathione reductase were important for redox homeostasis. Thioredoxin reductase contributed less during exponential phase when there was a strong requirement for active Yap1p transcription factor, but was critical during stationary phase. The absence of ROS detoxification systems, such as catalases or superoxide dismutases, had a lesser effect on glutathione E(h), but a more pronounced effect on ROS levels and MMP. These results reflect the major shift in ROS generation as cells switch from fermentative to respiratory metabolism and also showed that there was not a strong correlation between ROS production, MMP and cellular redox environment. Heterogeneity was detected in populations of strains with compromised anti-oxidant defences, and as cells aged they shifted from one cell type with low ROS content to another with much higher intracellular ROS.

Complex cellular responses to reactive oxygen species.

Genome-wide analyses of yeast provide insight into cellular responses to reactive oxygen species (ROS). Many deletion mutants are sensitive to at least one ROS, but no one oxidant is representative of 'oxidative stress' despite the widespread use of a single compound such as H(2)O(2). This has major implications for studies of pathological situations. Cells have a range of mechanisms for maintaining resistance that involves either induction or repression of many genes and extensive remodeling of the transcriptome. Cells have constitutive defense systems that are largely unique to each oxidant, but overlapping, inducible repair systems. The pattern of the transcriptional response to a particular ROS depends on its concentration, and 'classical' antioxidant systems that are induced by high concentrations of ROS can be repressed when cells adapt to low concentrations of ROS.

Footprinting the 'essential regulatory region' of the retinoblastoma gene promoter in intact human cells.

The retinoblastoma tumour suppressor protein is a key cell cycle regulator. Protein-DNA interactions at the retinoblastoma (RB1) promoter, including the 'essential regulatory region', were investigated using novel DNA-targeted nitrogen mustards in intact human cells. The footprinting experiments were carried out in two different environments: in intact HeLa and K562 cells where the access of DNA-targeted probes to chromatin is affected by cellular protein-DNA interactions associated with gene regulation; and in purified DNA where their access is unencumbered by protein-DNA interactions. Using the ligation-mediated PCR (LMPCR) technique, the sites of damage were determined at base pair resolution on DNA sequencing gels. Our results demonstrate that, in intact cells, footprints were observed at the E2F, ATF and RBF1/Sp1 DNA binding motifs in the RB1 promoter. In addition, a novel footprint was observed at a previously unidentified cycle homology region (CHR) and at four uncharacterised protein-DNA binding sites. In further experiments, nitrogen mustard-treated cells were FACS sorted into G1, S and G2/M phases of the cell cycle prior to LMPCR analysis. Expression of the RB1 gene is cell cycle-regulated and footprinting studies of the promoter in FACS-sorted cells indicated that transcription factor binding at the GC box, CHR binding motif and the 'essential regulatory region' are cell cycle dependent.

Genome-wide transcriptional responses to a lipid hydroperoxide: adaptation occurs without induction of oxidant defenses.

Free radicals can initiate the oxidation of polyunsaturated fatty acids in cells through the process of lipid peroxidation. The genome-wide transcriptional changes in Saccharomyces cerevisiae after treatment with the toxic lipid peroxidation product linoleic acid hydroperoxide (LoaOOH) were identified. High-dose treatment led to a switch in transcription from biosynthetic to protective functions. This response encompassed a set of genes stimulated predominantly by LoaOOH, and not by other oxidants or heat shock, which contained components of the pleiotropic drug resistance system. The dose dependence of the transcriptional response revealed that large and widespread changes occur only in response to higher doses. Pretreatment of cells with sublethal doses of LoaOOH induces resistance to an otherwise lethal dose through the process of adaptation. Adaptive doses elicited a more subtle transcriptional response affecting metabolic functions, including an increase in the capacity for detoxification and downregulation of the rate of protein synthesis. Surprisingly, the cellular response to adaptive doses did not include induction of oxidative-stress defense enzymes nor of transcripts involved in general cellular defense systems.

Genomic and phylogenetic footprinting at the epsilon-globin silencer region in intact human cells.

Chromatin structure at the silencer region of the epsilon-globin promoter was investigated using novel nitrogen mustards as probes of protein--DNA interactions. Sites of protection and enhancement that corresponded to known transcription factor binding sites were detected in both K562 and HeLa cells at this gene region. Protection was observed at several sites including the GATA-1/YY1 motifs. Of particular interest was a large 155 bp footprint that was observed at the epsilon-globin gene silencer region of the promoter. This large footprint was consistent with the presence of a positioned nucleosome core in intact human cells at this silencer region. Additionally, the DNA sequence at the epsilon-globin silencer and promoter was compared for 11 mammalian species. Significant areas of conservation were found that correlated with known transcription factor binding motifs. This phylogenetic footprinting analysis was compared to the genomic footprinting data at the epsilon-globin silencer region.

The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation.

Controlled production of the cytokine IL-2 plays a key role in the mammalian immune system. Expression from the gene is tightly regulated with no detectable expression in resting T cells and a strong induction following T cell activation. The IL-2 proximal promoter (+1 to -300) contains many well-defined transcriptional activation elements that respond to T cell stimulation. To determine the role of chromatin structure in the regulation of interleukin-2 gene transcription, nucleosome assembly across the IL-2 promoter region was examined using in vitro chromatin reconstitution assays. The IL-2 promoter assembles a nucleosome that is both translationally and rotationally positioned, spanning some of the major functional control elements. The binding of transcription factors to these elements, with the exception of the architectural protein HMGA1, was occluded by the presence of the nucleosome. Analysis of the chromatin architecture of the IL-2 gene in Jurkat T cells provided evidence for the presence of a similarly positioned nucleosome in vivo. The region encompassed by this nucleosome becomes remodeled following activation of Jurkat T cells. These observations suggest that the presence of a positioned nucleosome across the IL-2 proximal promoter may play an important role in maintaining an inactive gene in resting T cells and that remodeling of this nucleosome is important for gene activation.

The interaction of DNA-targeted 9-aminoacridine-4-carboxamide platinum complexes with DNA in intact human cells.

As part of an ongoing drug development programme, this paper describes the sequence specificity and time course of DNA adduct formation for a series of novel DNA-targeted analogues of cis-diaminedichloroplatinum(II) (cisplatin) (9-aminoacridine-4-carboxamide Pt complexes) in intact HeLa cells. The sequence specificity of DNA damage caused by cisplatin and analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification/polymerase stop assay. Primer extension is inhibited by a Pt-DNA adduct, and hence the sites of these lesions can be analysed on DNA sequencing gels. The repetitive alphoid DNA sequence was used as the target DNA in human cells. The 9-aminoacridine-4-carboxamide Pt complexes exhibited a markedly different sequence specificity relative to cisplatin and other analogues. The sequence specificity of the 9-aminoacridine-4-carboxamide Pt complexes is shifted away from a preference for runs of guanines. The 9-aminoacridine-4-carboxamide Pt complexes have an enhanced preference for GA dinucleotides. This is the first occasion that an altered DNA sequence specificity has been demonstrated for a cisplatin analogue in human cells. A time course of DNA damage revealed that the DNA-targeted Pt complexes, consisting of four 9-aminoacridine-4-carboxamide Pt complexes and one acridine-4-carboxamide Pt complex, damaged DNA more rapidly compared to cisplatin and non-targeted analogues. A comparison of the time taken to reach half the maximum relative intensity indicated that the DNA-targeted Pt complexes reacted approximately 4-fold faster than cisplatin and the non-targeted analogues.