Nucleic Acids Research

Su, Jie; Baigude, Huricha; McCarroll, Joshua; Rana, Tariq M.

doi: 10.1093/nar/gkq1307pmid: 21212128

MicroRNAs (miRNAs) are small endogenous non-coding RNAs that regulate post-transcriptional gene expression and are important in many biological processes. Disease-associated miRNAs have been shown to become potential targets for therapeutic intervention. Functions of miRNAs can be inhibited by using antisense oligonucleotides, called anti-miRs, complimentary to the miRNA sequences. Here, we show that systemic delivery of a chemically stabilized anti-miR-122 complexed with interfering nanoparticles (iNOPs) effectively silences the liver-expressed miR-122 in mice. Intravenous administration of 2mgkg1 chemically modified anti-miR-122 complexed with iNOP-7 resulted in 83.23.2 specific silencing of miR-122, which was accompanied by regulating gene expression in liver and lowering of plasma cholesterol. The specific silencing of miR-122 was long lasting and did not induce an immune response. Our results demonstrate that iNOPs can successfully deliver anti-miR to specifically target and silence miRNA in clinically acceptable and therapeutically affordable doses.

Fiston-Lavier, Anna-Sophie; Carrigan, Matthew; Petrov, Dmitri A.; Gonzlez, Josefa

doi: 10.1093/nar/gkq1291pmid: 21177644

Transposable elements (TEs) are repetitive DNA sequences that are ubiquitous, extremely abundant and dynamic components of practically all genomes. Much effort has gone into annotation of TE copies in reference genomes. The sequencing cost reduction and the newly available next-generation sequencing (NGS) data from multiple strains within a species offer an unprecedented opportunity to study population genomics of TEs in a range of organisms. Here, we present a computational pipeline (T-lex) that uses NGS data to detect the presence/absence of annotated TE copies. T-lex can use data from a large number of strains and returns estimates of population frequencies of individual TE insertions in a reasonable time. We experimentally validated the accuracy of T-lex detecting presence or absence of 768 previously identified TE copies in two resequenced Drosophila melanogaster strains. Approximately 95 of the TE insertions were detected with 100 sensitivity and 97 specificity. We show that even at low levels of coverage T-lex produces accurate results for TE copies that it can identify reliably but that the rate of no data calls increases as the coverage falls below 15. T-lex is a broadly applicable and flexible tool that can be used in any genome provided the availability of the reference genome, individual TE copy annotation and NGS data.

Ferraris, L.; Stewart, A. P.; Gemberling, M. P.; Reid, D. C.; Lapadula, M. J.; Thompson, W. A.; Fairbrother, W. G.

doi: 10.1093/nar/gkq1213pmid: 21169336

There are a variety of in vivo and in vitro methods to determine the genome-wide specificity of a particular trans-acting factor. However there is an inherent limitation to these candidate approaches. Most biological studies focus on the regulation of particular genes, which are bound by numerous unknown trans-acting factors. Therefore, most biological inquiries would be better addressed by a method that maps all trans-acting factors that bind particular regions rather than identifying all regions bound by a particular trans-acting factor. Here, we present a high-throughput binding assay that returns thousands of unbiased measurements of complex formation on nucleic acid. We applied this method to identify transcriptional complexes that form on DNA regions upstream of genes involved in pluripotency in embryonic stem cells (ES cells) before and after differentiation. The raw binding scores, motif analysis and expression data are used to computationally reconstruct remodeling events returning the identity of the transcription factor(s) most likely to comprise the complex. The most significant remodeling event during ES cell differentiation occurred upstream of the REST gene, a transcriptional repressor that blocks neurogenesis. We also demonstrate how this method can be used to discover RNA elements and discuss applications of screening polymorphisms for allelic differences in binding.

doi: 10.1093/nar/gkr212pmid: N/A

Li, Jun; Zhang, Jia-Min; Li, Xin; Suo, Fang; Zhang, Mei-Jun; Hou, Wenru; Han, Jinghua; Du, Li-Lin

doi: 10.1093/nar/gkq1358pmid: 21247877

The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis tool in various eukaryotic organisms. Here, we show that PB transposes in the fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We developed a PB-based mutagenesis system for S. pombe by constructing a strain with a selectable transposon excision marker and an integrated transposase gene. PB transposition in this strain has low chromosomal distribution bias as shown by deep sequencing-based insertion site mapping. Using this system, we obtained loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1 from a screen for mutants resistant to the microtubule-destabilizing drug thiabendazole. From another screen for cdc25-22 suppressors, we obtained multiple alleles of wee1 as expected. The success of these two screens demonstrated the usefulness of this PB-mediated mutagenesis tool for fission yeast.

Ye, Tao; Krebs, Arnaud R.; Choukrallah, Mohamed-Amin; Keime, Celine; Plewniak, Frederic; Davidson, Irwin; Tora, Laszlo

doi: 10.1093/nar/gkq1287pmid: 21177645

In a single experiment, chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq) provides genome-wide information about a given covalent histone modification or transcription factor occupancy. However, time efficient bioinformatics resources for extracting biological meaning out of these gigabyte-scale datasets are often a limiting factor for data interpretation by biologists. We created an integrated portable ChIP-seq data interpretation platform called seqMINER, with optimized performances for efficient handling of multiple genome-wide datasets. seqMINER allows comparison and integration of multiple ChIP-seq datasets and extraction of qualitative as well as quantitative information. seqMINER can handle the biological complexity of most experimental situations and proposes methods to the user for data classification according to the analysed features. In addition, through multiple graphical representations, seqMINER allows visualization and modelling of general as well as specific patterns in a given dataset. To demonstrate the efficiency of seqMINER, we have carried out a comprehensive analysis of genome-wide chromatin modification data in mouse embryonic stem cells to understand the global epigenetic landscape and its change through cellular differentiation.

doi: 10.1093/nar/gkr211pmid: N/A

doi: 10.1093/nar/gkr210pmid: N/A

Wang, Lei; Zhang, Yingwei; Tian, Jingqi; Li, Hailong; Sun, Xuping

doi: 10.1093/nar/gkq1294pmid: 21183465

In this article, we report on the facile and rapid synthesis of conjugation polymer poly(p-phenylenediamine) nanobelts (PNs) via room temperature chemical oxidation polymerization of p-phenylenediamine monomers by ammonium persulfate in aqueous medium. We further demonstrate the proof-of-concept that PNs can be used as an effective fluorescent sensing platform for nucleic acid detection for the first time. The general concept used in this approach lies in the facts that the adsorption of the fluorescently labeled single-stranded DNA probe by PN leads to substantial fluorescence quenching, followed by specific hybridization with the complementary region of the target DNA sequence. This results in desorption of the hybridized complex from PN surface and subsequent recovery of fluorescence. We also show that the sensing platform described herein can be used for multiplexing detection of nucleic acid sequences.

Nagarajan, Niranjan; Kingsford, Carl

doi: 10.1093/nar/gkq1232pmid: 21177643

Reassortments in the influenza virusa process where strains exchange genetic segmentshave been implicated in two out of three pandemics of the 20th century as well as the 2009 H1N1 outbreak. While advances in sequencing have led to an explosion in the number of whole-genome sequences that are available, an understanding of the rate and distribution of reassortments and their role in viral evolution is still lacking. An important factor in this is the paucity of automated tools for confident identification of reassortments from sequence data due to the challenges of analyzing large, uncertain viral phylogenies. We describe here a novel computational method, called GiRaF (Graph-incompatibility-based Reassortment Finder), that robustly identifies reassortments in a fully automated fashion while accounting for uncertainties in the inferred phylogenies. The algorithms behind GiRaF search large collections of Markov chain Monte Carlo (MCMC)-sampled trees for groups of incompatible splits using a fast biclique enumeration algorithm coupled with several statistical tests to identify sets of taxa with differential phylogenetic placement. GiRaF correctly finds known reassortments in human, avian, and swine influenza populations, including the evolutionary events that led to the recent swine flu outbreak. GiRaF also identifies several previously unreported reassortments via whole-genome studies to catalog events in H5N1 and swine influenza isolates.