| 1. |
- Chalk, Alistair
(författare)
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Computational prediction of antisense oligonucleotides and siRNAs
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Two popular gene knockdown methods, antisense oligonucleotides (AOs) and short interfering RNAs (siRNAs) are commonly used to selectively inhibit gene expression (gene knockdown). This is an extremely valuable tool for functional genomics, however the selection of effective molecules using either method is non trivial. Here we present a number of computational solutions including predictive methods for AO and siRNA molecules, a database of siRNAs of known efficacy and a visualization tool for viewing complex sequence analysis scenarios. Prediction models for AOs based on the machine learning methods Artificial Neural Networks (AOpredict, paper I) and Support Vector Machines (SVMPredict, paper II) address the problem of AO design by applying computational methods to a database of AOs mined from the literature. These models predict AO efficacy at high accuracy according to cross-validation results. A database of siRNAs was created from the literature and contains 1276 siRNAs targeting 116 genes; this database is available through a web interface (siRNAdb, paper III). The properties of functional and non-functional siRNAs were examined, current siRNA design rules were evaluated and a new prediction method was developed (siSearch, paper IV). The issue of off-target effects was examined in detail and a scoring scheme was developed based on the available experimental data (SpecificityServer, paper V). We identified that 14-23% of siRNAs in the database are likely to elicit off-target effects. A tool for viewing multiple series of biological information and computational predictions was developed to view data from SFS and GFF formats, simplifying the analysis process (Sfixem, paper VI). In an independent validation of 63 siRNAs designed by siSearch to target 31 Rat genes, 19 (30%) had a silencing activity of 90% or higher and 3 3 (52%) silenced to a level of 70% or more. For 25 of the 31 genes siSearch was successful in predicting siRNAs that silence the gene by more than 80%. The prediction methods and specificity server, all of which are available online are highly applicable tools for die practical design of high efficacy specific siRNAs and AOs.
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| 2. |
- Chalk, Alistair M, et al.
(författare)
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Gene expression profiling to define the cell intrinsic role of the SKI proto-oncogene in hematopoiesis and myeloid neoplasms.
- 2014
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Ingår i: Genomics Data. - : Elsevier BV. - 2213-5960. ; 2, s. 189-191
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Tidskriftsartikel (refereegranskat)abstract
- The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced gene signatures associated with hematopoietic stem cells and myeloid differentiation. Here we provide detailed experimental methods and analysis for the gene expression profiling described in our recently published study of Singbrant et al. (2014) in Haematologica. Our data sets (available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39457) provide a resource for exploring the underlying molecular mechanisms of the involvement of the proto-oncogene SKI in hematopoietic stem cell function and development of myeloid neoplasms.
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| 3. |
- Chalk, Alistair M, et al.
(författare)
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siRNA specificity searching incorporating mismatch tolerance data.
- 2008
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1460-2059. ; 24:10, s. 1316-1317
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Tidskriftsartikel (refereegranskat)abstract
- Artificially synthesized short interfering RNAs (siRNAs) are widely used in functional genomics to knock down specific target genes. One ongoing challenge is to guarantee that the siRNA does not elicit off-target effects. Initial reports suggested that siRNAs were highly sequence-specific; however, subsequent data indicates that this is not necessarily the case. It is still uncertain what level of similarity and other rules are required for an off-target effect to be observed, and scoring schemes have not been developed to look beyond simple measures such as the number of mismatches or the number of consecutive matching bases present. We created design rules for predicting the likelihood of a non-specific effect and present a web server that allows the user to check the specificity of a given siRNA in a flexible manner using a combination of methods. The server finds potential off-target matches in the corresponding RefSeq database and ranks them according to a scoring system based on experimental studies of specificity. AVAILABILITY: The server is available at http://informatics-eskitis.griffith.edu.au/SpecificityServer.
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| 4. |
- Hong, Junmei, et al.
(författare)
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Focusing on RISC assembly in mammalian cells.
- 2008
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Ingår i: Biochem Biophys Res Commun. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 368:3, s. 703-8
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5' end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5' end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.
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| 5. |
- Singbrant, Sofie, et al.
(författare)
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The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease
- 2014
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 99:4, s. 647-655
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Tidskriftsartikel (refereegranskat)abstract
- The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.
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