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  • Journals
  • Articles  (4)
  • RNA characterisation and manipulation
  • Oxford University Press  (4)
  • American Chemical Society (ACS)
  • Frontiers Media
  • PeerJ
  • 2015-2019  (4)
  • 1985-1989
  • 1980-1984
  • 2016  (4)
  • Biology  (4)
  • Nature of Science, Research, Systems of Higher Education, Museum Science
  • Education
Collection
  • Journals
  • Articles  (4)
Publisher
  • Oxford University Press  (4)
  • American Chemical Society (ACS)
  • Frontiers Media
  • PeerJ
Years
  • 2015-2019  (4)
  • 1985-1989
  • 1980-1984
Year
Topic
  • Biology  (4)
  • Nature of Science, Research, Systems of Higher Education, Museum Science
  • Education
  • 1
    Publication Date: 2016-06-03
    Description: N 6 -methyladenosine (m 6 A) is a prevalent RNA methylation modification involved in the regulation of degradation, subcellular localization, splicing and local conformation changes of RNA transcripts. High-throughput experiments have demonstrated that only a small fraction of the m 6 A consensus motifs in mammalian transcriptomes are modified. Therefore, accurate identification of RNA m 6 A sites becomes emergently important. For the above purpose, here a computational predictor of mammalian m 6 A site named SRAMP is established. To depict the sequence context around m 6 A sites, SRAMP combines three random forest classifiers that exploit the positional nucleotide sequence pattern, the K-nearest neighbor information and the position-independent nucleotide pair spectrum features, respectively. SRAMP uses either genomic sequences or cDNA sequences as its input. With either kind of input sequence, SRAMP achieves competitive performance in both cross-validation tests and rigorous independent benchmarking tests. Analyses of the informative features and overrepresented rules extracted from the random forest classifiers demonstrate that nucleotide usage preferences at the distal positions, in addition to those at the proximal positions, contribute to the classification. As a public prediction server, SRAMP is freely available at http://www.cuilab.cn/sramp/ .
    Keywords: RNA characterisation and manipulation
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 2
    Publication Date: 2016-04-08
    Description: Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant ( k cat ) of a model ribozyme.
    Keywords: RNA characterisation and manipulation
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 3
    Publication Date: 2016-12-04
    Description: Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a U A G premature termination codon (PTC) to tryptophan (U G G) was improved from ~11 % to ~70 %. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.
    Keywords: RNA characterisation and manipulation
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 4
    Publication Date: 2016-09-20
    Description: RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from Bacteria, Archaea and Eukarya. RNAs bearing 2'-O-methylations show increased resistance to degradation and enhanced stability in helices. While the exact role of each 2'-O-Me residue remained elusive, the catalytic protein Fibrillarin (Nop1 in yeast) responsible for 2'-O-methylation in eukaryotes, is associated with human pathologies. Therefore, there is an urgent need to precisely map and quantify hundreds of 2'-O-Me residues in RNA using high-throughput technologies. Here, we develop a reliable protocol using alkaline fragmentation of total RNA coupled to a commonly used ligation approach, and Illumina sequencing. We describe a methodology to detect 2'-O-methylations with high sensitivity and reproducibility even with limited amount of starting material (1 ng of total RNA). The method provides a quantification of the 2'-O-methylation occupancy of a given site, allowing to detect relatively small changes (〉10%) in 2'-O-methylation profiles. Altogether this technique unlocks a technological barrier since it will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2'-O-methylations in pathologies.
    Keywords: RNA characterisation and manipulation
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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