Experimental validation of the importance of seed complement frequency to siRNA specificity

Anderson, Emily M.; Birmingham, Amanda; Baskerville, Scott; Reynolds, Angela; Maksimova, Elena; Leake, Devin; Fedorov, Yuriy; Karpilow, Jon; Khvorova, Anastasia

doi:10.1261/rna.704708

Cited by 135 publications

(128 citation statements)

References 32 publications

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“…To eliminate the characterization of artifacts arising through off-target effects, 3 independent FEN1 silencing conditions were used-2 independent siRNA duplexes (FEN1-2 and FEN1-3) and a FEN1-pool comprised of 4 independent siRNA duplexes (effectively quartering the concentration of each duplex), that has been shown to greatly diminish off-target effects (42)(43)(44)(45)(46). Using fixed and live cell HC-DIM, we showed that diminished FEN1 expression adversely affects overall cell numbers, which presumably occurs through the corresponding increases in cellular cytotoxicity.…”

Section: Discussionmentioning

confidence: 99%

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing

McManus

Barrett

Nouhi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

121

131

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Mutations that cause chromosome instability (CIN) in cancer cells produce ''sublethal'' deficiencies in an essential process (chromosome segregation) and, therefore, may represent a major untapped resource that could be exploited for therapeutic benefit in the treatment of cancer. If second-site unlinked genes can be identified, that when knocked down, cause a synthetic lethal (SL) phenotype in combination with a somatic mutation in a CIN gene, novel candidate therapeutic targets will be identified. To test this idea, we took a cross species SL candidate gene approach by recapitulating a SL interaction observed between rad54 and rad27 mutations in yeast, via knockdown of the highly sequence-and functionally-related proteins RAD54B and FEN1 in a cancer cell line. We show that knockdown of RAD54B, a gene known to be somatically mutated in cancer, causes CIN in mammalian cells. Using high-content microscopy techniques, we demonstrate that RAD54B-deficient human colorectal cancer cells are sensitive to SL killing by reduced FEN1 expression, while isogenic RAD54B proficient cells are not. This conserved SL interaction suggests that extrapolating SL interactions observed in model organisms for homologous genes mutated in human cancers will aid in the identification of novel therapeutic targets for specific killing of cancerous cells exhibiting CIN.cancer therapeutics ͉ chromosome instability ͉ synthetic lethality

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Section: Discussionmentioning

confidence: 99%

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing

McManus

Barrett

Nouhi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

121

131

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“…This can be, for example, influenced by the seed binding energy and seed composition, which would determine the pool of potential binding sites in the transcriptome and the difference between on-target and off-target RNAs (Das et al, 2013a;Das et al, 2013b). Adaptations of siRNA/shRNA design to reduce off-target effects include weak base pairing in both seed and 3 ' regions and evaluation of potential cross-hybridization candidates (Yamada and Morishita, 2005;Anderson et al, 2008). Reduced off-targeting features were subsequently integrated into siRNA design tools such as siDirect (Naito et al, 2009;Naito and Ui-Tei, 2013).…”

Section: Better Small Rna Designmentioning

confidence: 99%

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

Pačes

Nič²,

Novotný³

et al. 2017

EFS3

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This report is the outcome of an EFSA procurement aiming at investigating and summarising the state of knowledge on (I) the mode-of-action of dsRNA and miRNA pathways, (II) the potential for nontarget gene regulation by dsRNA-derived siRNAs or miRNAs, (III) the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing. The report is based on a comprehensive and systematic literature search, starting with the identification and retrieval of~190,000 publications related to the research area and further filtered down with keywords to produce focused collections used for subsequent screening of titles and abstracts. The report is comprised of an (I) Introduction to the field of small RNAs, (II) a Data and Methodologies section containing strategies used for literature search and study selection, and (III) the Results of the literature review organized according to the three main procurement tasks. The outcome of the first task reviews dsRNA and miRNA pathways in mammals (including humans), birds, fish, arthropods, annelids, molluscs, nematodes, and plants. Eight taxon-dedicated chapters are based on~1,400 cumulative references chosen from~10,000 inspected titles and abstracts. We review conserved and divergent aspects of small RNA pathways and dsRNA responses in animals and plants including structure and function of key proteins as well as four basic mechanisms: genome-encoded posttranscriptional regulations (miRNA), degradation of RNAs by short interfering RNA pools generated from long dsRNA (RNAi), transcriptional silencing, and sequence-independent responses to dsRNA. The outcome of the second task focuses on base pairing between small RNAs and their target RNAs and predictability of biological effects of small RNAs in animals and plants. The outcome of the last task reviews methodology, siRNA pools, and movement of small RNAs in plants. Potential transfer of small RNAs between species and circulating miRNAs in mammals is described in the final chapter.

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“…These siRNAs consist of four pooled sequences rationally designed to minimize off-target effects [13][14][15]. The ON-TARGETplus™ Human SMARTpool siRNA sequences used were PMCA2 (siPMCA2, L-006116-00) and the non-targeting control siRNA (siNT, D-001810-10).…”

Section: Rna Interferencementioning

confidence: 99%

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

Curry

Roberts‐Thomson

Monteith

2016

Biochemical and Biophysical Research Communications

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PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin.Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway.

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Experimental validation of the importance of seed complement frequency to siRNA specificity

Cited by 135 publications

References 32 publications

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

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