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  • 1
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    tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers (2016)
    Oxford University Press
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    Publication Date: 2016-07-06
    Description: tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called ‘tRFinCancer’ was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called ‘tRFBrowser’ shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/ .
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion (2015)
    Oxford University Press
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    Publication Date: 2015-06-24
    Description: With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure–function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus–host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.
    Keywords: Cell biology, DNA-Mediated Cell Transformation and Nucleic Acids Transfer
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    A systematic survey of the Cys2His2 zinc finger DNA-binding landscape (2015)
    Oxford University Press
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    Publication Date: 2015-02-18
    Description: Cys 2 His 2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of 〉160 000 unique domain–DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain–DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA (2014)
    Oxford University Press
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    Publication Date: 2014-10-10
    Description: RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp- d -Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvβ3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvβ3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    An intensity ratio of interlocking loops determines circadian period length (2014)
    Oxford University Press
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    Publication Date: 2014-09-17
    Description: Circadian clocks allow organisms to orchestrate the daily rhythms in physiology and behaviors, and disruption of circadian rhythmicity can profoundly affect fitness. The mammalian circadian oscillator consists of a negative primary feedback loop and is associated with some ‘auxiliary’ loops. This raises the questions of how these interlocking loops coordinate to regulate the period and maintain its robustness. Here, we focused on the REV-ERBα/ Cry1 auxiliary loop, consisting of Rev-Erbα/ROR-binding elements (RORE) mediated Cry1 transcription, coordinates with the negative primary feedback loop to modulate the mammalian circadian period. The silicon simulation revealed an unexpected rule: the intensity ratio of the primary loop to the auxiliary loop is inversely related to the period length, even when post-translational feedback is fixed. Then we measured the mRNA levels from two loops in 10-mutant mice and observed the similar monotonic relationship. Additionally, our simulation and the experimental results in human osteosarcoma cells suggest that a coupling effect between the numerator and denominator of this intensity ratio ensures the robustness of circadian period and, therefore, provides an efficient means of correcting circadian disorders. This ratio rule highlights the contribution of the transcriptional architecture to the period dynamics and might be helpful in the construction of synthetic oscillators.
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 6
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    Error removal in microchip-synthesized DNA using immobilized MutS (2014)
    Oxford University Press
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    Publication Date: 2014-07-04
    Description: The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ~21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    SpermatogenesisOnline 1.0: a resource for spermatogenesis based on manual literature curation and genome-wide data mining (2012)
    Oxford University Press
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    Publication Date: 2012-12-20
    Description: Human infertility affects 10–15% of couples, half of which is attributed to the male partner. Abnormal spermatogenesis is a major cause of male infertility. Characterizing the genes involved in spermatogenesis is fundamental to understand the mechanisms underlying this biological process and in developing treatments for male infertility. Although many genes have been implicated in spermatogenesis, no dedicated bioinformatic resource for spermatogenesis is available. We have developed such a database, SpermatogenesisOnline 1.0 ( http://mcg.ustc.edu.cn/sdap1/spermgenes/ ), using manual curation from 30 233 articles published before 1 May 2012. It provides detailed information for 1666 genes reported to participate in spermatogenesis in 37 organisms. Based on the analysis of these genes, we developed an algorithm, Greed AUC Stepwise (GAS) model, which predicted 762 genes to participate in spermatogenesis (GAS probability 〉0.5) based on genome-wide transcriptional data in Mus musculus testis from the ArrayExpress database. These predicted and experimentally verified genes were annotated, with several identical spermatogenesis-related GO terms being enriched for both classes. Furthermore, protein–protein interaction analysis indicates direct interactions of predicted genes with the experimentally verified ones, which supports the reliability of GAS. The strategy (manual curation and data mining) used to develop SpermatogenesisOnline 1.0 can be easily extended to other biological processes.
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    Topics: Biology
    Published by Oxford University Press
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  • 8
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    Probing DNA clamps with single-molecule force spectroscopy (2013)
    Oxford University Press
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    Publication Date: 2013-09-06
    Description: Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.
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    Topics: Biology
    Published by Oxford University Press
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  • 9
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    Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method (2013)
    Oxford University Press
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    Publication Date: 2013-04-14
    Description: The genome-wide distribution patterns of the ‘6th base’ 5-hydroxymethylcytosine (5hmC) in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, it has been challenging to directly compare different data sets and samples using data generated by this method. Here, we report a new comparative hMeDIP-seq method, which involves barcoding different input DNA samples at the start and then performing hMeDIP-seq for multiple samples in one hMeDIP reaction. This approach extends the barcode technology from simply multiplexing the DNA deep sequencing outcome and provides significant advantages for quantitative control of all experimental steps, from unbiased hMeDIP to deep sequencing data analysis. Using this improved method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESC-derived neural progenitor cells (NPCs). We identified differentially hydroxymethylated regions (DHMRs) between ESCs and NPCs and uncovered an intricate relationship between the alteration of DNA hydroxymethylation and changes in gene expression during neural lineage commitment of ESCs. Presumably, the DHMRs between ESCs and NPCs uncovered by this approach may provide new insight into the function of 5hmC in gene regulation and neural differentiation. Thus, this newly developed comparative hMeDIP-seq method provides a cost-effective and user-friendly strategy for direct genome-wide comparison of DNA hydroxymethylation across multiple samples, lending significant biological, physiological and clinical implications.
    Keywords: Massively Parallel (Deep) Sequencing, Chromatin and Epigenetics
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    Topics: Biology
    Published by Oxford University Press
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  • 10
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    COME: a robust coding potential calculation tool for lncRNA identification and characterization based on multiple features (2017)
    Oxford University Press
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    Publication Date: 2017-01-10
    Description: Recent genomic studies suggest that novel long non-coding RNAs (lncRNAs) are specifically expressed and far outnumber annotated lncRNA sequences. To identify and characterize novel lncRNAs in RNA sequencing data from new samples, we have developed COME, a co ding potential calculation tool based on m ultiple f e atures. It integrates multiple sequence-derived and experiment-based features using a decompose–compose method, which makes it more accurate and robust than other well-known tools. We also showed that COME was able to substantially improve the consistency of predication results from other coding potential calculators. Moreover, COME annotates and characterizes each predicted lncRNA transcript with multiple lines of supporting evidence, which are not provided by other tools. Remarkably, we found that one subgroup of lncRNAs classified by such supporting features (i.e. conserved local RNA secondary structure) was highly enriched in a well-validated database (lncRNAdb). We further found that the conserved structural domains on lncRNAs had better chance than other RNA regions to interact with RNA binding proteins, based on the recent eCLIP-seq data in human, indicating their potential regulatory roles. Overall, we present COME as an accurate, robust and multiple-feature supported method for the identification and characterization of novel lncRNAs. The software implementation is available at https://github.com/lulab/COME .
    Keywords: RNA characterisation and manipulation, Computational Methods
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    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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