siDirect 2.0: updated software for designing functional siRNA with reduced seed-dependent off-target effect

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi), mediated by 21-nucleotide (nt)-length small interfering RNAs (siRNAs), is a powerful tool not only for studying gene function but also for therapeutic applications. RNAi, requiring perfect complementarity between the siRNA guide strand and the target mRNA, was believed to be extremely specific. However, a recent growing body of evidence has suggested that siRNA could down-regulate unintended genes whose transcripts possess complementarity to the 7-nt siRNA seed region. This off-target gene silencing may often provide incongruous results obtained from knockdown experiments, leading to misinterpretation. Thus, an efficient algorithm for designing functional siRNAs with minimal off-target effect based on the mechanistic features is considered of value.</p> <p>Results</p> <p>We present siDirect 2.0, an update of our web-based software siDirect, which provides functional and off-target minimized siRNA design for mammalian RNAi. The previous version of our software designed functional siRNAs by considering the relationship between siRNA sequence and RNAi activity, and provided them along with the enumeration of potential off-target gene candidates by using a fast and sensitive homology search algorithm. In the new version, the siRNA design algorithm is extensively updated to eliminate off-target effects by reflecting our recent finding that the capability of siRNA to induce off-target effect is highly correlated to the thermodynamic stability, or the melting temperature (Tm), of the seed-target duplex, which is formed between the nucleotides positioned at 2-8 from the 5' end of the siRNA guide strand and its target mRNA. Selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm < 21.5°C) followed by the elimination of unrelated transcripts with nearly perfect match should minimize the off-target effects.</p> <p>Conclusion</p> <p>siDirect 2.0 provides functional, target-specific siRNA design with the updated algorithm which significantly reduces off-target silencing. When the candidate functional siRNAs could form seed-target duplexes with Tm values below 21.5°C, and their 19-nt regions spanning positions 2-20 of both strands have at least two mismatches to any other non-targeted transcripts, siDirect 2.0 can design at least one qualified siRNA for >94% of human mRNA sequences in RefSeq. siDirect 2.0 is available at <url>http://siDirect2.RNAi.jp/</url>.</p

Selecting effective siRNA sequences by using radial basis function network and decision tree learning

BACKGROUND: Although short interfering RNA (siRNA) has been widely used for studying gene functions in mammalian cells, its gene silencing efficacy varies markedly and there are only a few consistencies among the recently reported design rules/guidelines for selecting siRNA sequences effective for mammalian genes. Another shortcoming of the previously reported methods is that they cannot estimate the probability that a candidate sequence will silence the target gene. RESULTS: We propose two prediction methods for selecting effective siRNA target sequences from many possible candidate sequences, one based on the supervised learning of a radial basis function (RBF) network and other based on decision tree learning. They are quite different from the previous score-based siRNA design techniques and can predict the probability that a candidate siRNA sequence will be effective. The proposed methods were evaluated by applying them to recently reported effective and ineffective siRNA sequences for various genes (15 genes, 196 siRNA sequences). We also propose the combined prediction method of the RBF network and decision tree learning. As the average prediction probabilities of gene silencing for the effective and ineffective siRNA sequences of the reported genes by the proposed three methods were respectively 65% and 32%, 56.6% and 38.1%, and 68.5% and 28.1%, the methods imply high estimation accuracy for selecting candidate siRNA sequences. CONCLUSION: New prediction methods were presented for selecting effective siRNA sequences. As the proposed methods indicated high estimation accuracy for selecting candidate siRNA sequences, they would be useful for many other genes

A multivariate prediction model for microarray cross-hybridization

BACKGROUND: Expression microarray analysis is one of the most popular molecular diagnostic techniques in the post-genomic era. However, this technique faces the fundamental problem of potential cross-hybridization. This is a pervasive problem for both oligonucleotide and cDNA microarrays; it is considered particularly problematic for the latter. No comprehensive multivariate predictive modeling has been performed to understand how multiple variables contribute to (cross-) hybridization. RESULTS: We propose a systematic search strategy using multiple multivariate models [multiple linear regressions, regression trees, and artificial neural network analyses (ANNs)] to select an effective set of predictors for hybridization. We validate this approach on a set of DNA microarrays with cytochrome p450 family genes. The performance of our multiple multivariate models is compared with that of a recently proposed third-order polynomial regression method that uses percent identity as the sole predictor. All multivariate models agree that the 'most contiguous base pairs between probe and target sequences,' rather than percent identity, is the best univariate predictor. The predictive power is improved by inclusion of additional nonlinear effects, in particular target GC content, when regression trees or ANNs are used. CONCLUSION: A systematic multivariate approach is provided to assess the importance of multiple sequence features for hybridization and of relationships among these features. This approach can easily be applied to larger datasets. This will allow future developments of generalized hybridization models that will be able to correct for false-positive cross-hybridization signals in expression experiments

Telomerase inhibition, Telomere attrition and proliferation arrest of cancer cells induced by Phosphorothioate ASO-NLS conjugates targeting hTERC and siRNAs targeting hTERT

Telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. Recently, one avenue of cancer research focuses on antisense strategy to target the oncogenes or cancer driver genes, in a sequence specific fashion to down-regulate the expression of the target gene. The protein catalytic subunit, human telomerase reverse transcriptase (hTERT) and the template RNA component (hTERC) are essential for telomerase function, thus theoretically, inhibition of telomerase activity can be achieved by interfering with either the gene expression of hTERT or the hTERC of the telomerase enzymatic complex. The present study showed that phosphorothioate antisense oligonucleotide (sASO)-nuclear localization signal (NLS) peptide conjugates targeting hTERC could inhibit telomerase activity very efficiently at 5 μM concentration but less efficiently at 1 μM concentration. On the other hand, siRNA targeting hTERT mRNA could strongly suppress hTERT expression at 200 nM concentration. It was also revealed that siRNA targeting hTERT could induce telomere attrition and then irreversible arrest of proliferation of cancer cells

The Role of PP2A Phosphatase Activator (PTPA) in the biogenesis of PP2A in mammalian cells

Proteinphosphatase 2A (PP2A) ist eine essentielle Protein-Serin/Threonin Phosphatase und setzt sich aus einer katalytischen Untereinheit, einer strukturgebenden Untereinheit und einer von vielen regulatorischen Untereinheiten, die für Substratspezifität, subzelluläre Lokalisation und katalytische Aktivität verantwortlich sind, zusammen. Einer ungebundenen katalytischen Untereinheit fehlt die Substratspezifität, die sie erst durch Bindung an eine der vielen regulatorischen Untereinheiten erlangt. Freie und aktive katalytische Untereinheiten würden deshalb ein Risiko für Zellen darstellen. In einer vorhergehenden Studie im Labor von E. Ogris wurde im Modellorganismus Hefe nachgewiesen, dass die katalytische Untereinheit in inaktiver Form und mit veränderter Konformation vorliegen kann (Fellner et al., 2003). Die Aktivierung der katalytischen Untereinheit durch den Aktivator RRD (Rapamycin Resistant Deletion) ist dabei eng an den Aufbau des Holoenzyms gekoppelt. Das Thema dieser Arbeit war es, die Rolle von PTPA in der Biogenese von PP2A in einem Säugetier-Zellkultursystem zu studieren, indem die Expression von PTPA durch induzierbare RNAi gezielt supprimiert werden sollte.Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase and is a heterotrimer built up by a well-conserved catalytic subunit, a scaffolding subunit and one member out of a family of regulatory subunits, which determines the substrate specificity, (sub) cellular localization and catalytic activity of the PP2A. An activated monomer of the catalytic subunit would be unspecific and thus constitute a risk for the cell. In a previous study in yeast, it was shown that the catalytic subunit of PP2A is produced as a low active precursor that is activated by a protein named Rapamycin Resistant Deletion (RRD). Activation of PP2A by RRD is tightly linked to holoenzyme formation, thus providing a mechanism, preventing free and active catalytic subunit from harming the cell (Fellner et al., 2003). The subject of this work was to elucidate the role of PTPA in the biogenesis of PP2A in a mammalian cell culture system by suppression of PTPA expression by inducible RNA-interference

Potente HIV-gerichtete RNA-Interferenz mittels Selektion einer randomisierten lentiviralen shRNA-Bibliothek

Um neue potente shRNA-Sequenzen gegen HIV-1 zu identifizieren, wurde zum ersten Mal eine randomisierte lentivirale shRNA-Bibliothek konstruiert. Die Expression der shRNAs wurde dabei von einem RNA Polymerase III Promoter (Pol III) gesteuert. Die Selektion potenter shRNA-Sequenzen erfolgte über zu diesem Zweck etablierten Zelllinien. Sowohl die Konstruktion als auch das Selektionssystem basierten auf etablierten Technologien, die jedoch in einigen Punkten zwecks erhöhter Diversität der Bibiothek und optimierter Selektionsbedingungen modifiziert wurden. Die Validierung der selektierten shRNA-Sequenzen zeigte, dass mehr als die Hälfte der shRNAs ein inhibitorisches Potential von über 70% aufwiesen. Sequenzanalysen subklonierter shRNA-Expressionskassetten demonstrierten ihre HIV-1 Spezifität. Außerdem zeigten Vergleichsanalysen mit öffentlichen siRNA-Algorithmen, dass die meisten der hier selektierten shRNAs die Parameter der Algorithmen nicht erfüllen konnten. Daraus resultiert, dass die selektierten shRNA-Sequenzen zusammen mit anderen selektierten Bibliotheken einen wichtigen Beitrag zur Verbesserung der entscheidenden Parameter der siRNA-Algorithmen leisten könnten, weil sie aufgrund ihrer Effizienz und möglicher Toxizität und nicht ihrer Sequenz selektiert wurden. Um die Konvertibilität der inhibitorischen Sequenzen zu überprüfen, wurde das Pol III System mit synthetischen siRNAs und RNA Polymerase II gesteuerten miRNAs verglichen. Dabei stellte sich das Pol III System als das effizienteste heraus. Synthetische siRNA-Derivate ausgewählter hochpotenter shRNA-Sequenzen wiesen zwar ein vergleichbar starkes inhibitorisches Potential auf, die transiente Wirkdauer und die geringe Stabilität einiger siRNA-Sequenzen in Serum limitiert jedoch ihren Einsatz in klinischen Studien. Die Expression der shRNAs in einer miRNA-Umgebung offenbarte wiederum nur eine geringe inhibitorische Wirkung und erfordert weitere Optimierungen dieses Expressionssystems. Die stabile lentivirale Expression von shRNAs in HIV permissiven HeLa-P4-Zellen zeigte eine effiziente Inhibition der Virusreplikation nach Infektion mit HIV-1. Da die inserierten shRNAs in Zelllinien selektiert und reevaluiert wurden, kann außerdem von einer geringen Zytotoxizität ausgegangen werden. Dies macht sie für einen etwaigen Einsatz in gentherapeutischen Ansätzen interessant