Biomedical subjects

Angela Reynolds

Publications and source records attributed to Angela Reynolds.

11 recordsLinked to original sources

PubMed2006

Off-target effects by siRNA can induce toxic phenotype.

Although recent microarray studies have provided evidence of RNA interference (RNAi)-mediated off-target gene modulation, little is known about whether these changes induce observable phenotypic outcomes. Here we show that a fraction of randomly selected small inhibitory RNAs (siRNAs) can induce changes in cell viability in a target-independent fashion. The observed toxicity requires an intact RNAi pathway and can be eliminated by the addition of chemical modifications that reduce off-target effects. Furthermore, an analysis of toxic and nontoxic duplexes identifies a strong correlation between the toxicity and the presence of a 4-base-pair motif (UGGC) in the RISC-entering strand of toxic siRNA. This article provides further evidence of siRNA-induced off-target effects generating a measurable phenotype and also provides an example of how such undesirable phenotypes can be mitigated by addition of chemical modifications to the siRNA.

Base Sequence↗

PubMed2006

Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing.

Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended ("off-target") silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially complementary transcripts by all siRNAs tested. Silencing of perfectly matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2'-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position dependence of 2'-O-methyl ribosyl modification contrasts with the broader position dependence of base-pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts.

Base Sequence↗

PubMed2006

A reduced mathematical model of the acute inflammatory response II. Capturing scenarios of repeated endotoxin administration.

Bacterial lipopolysaccharide (LPS; endotoxin) is a potent immunostimulant that can induce an acute inflammatory response comparable to a bacterial infection. Experimental observations demonstrate that this biological response can be either blunted (tolerance) or augmented (potentiation) with repeated administration of endotoxin. Both phenomena are of clinical relevance. We show that a four-dimensional differential equation model of this response reproduces many scenarios involving repeated endotoxin administration. In particular, the model can display both tolerance and potentiation from a single parameter set, under different administration scenarios. The key determinants of the outcome of our simulations are the relative time-scales of model components. These findings support the hypothesis that endotoxin tolerance and other related phenomena can be considered as dynamic manifestations of a unified acute inflammatory response, and offer specific predictions related to the dynamics of this response to endotoxin.

Acute Disease↗

PubMed2006

Induction of the interferon response by siRNA is cell type- and duplex length-dependent.

Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell type-specific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.

Cell Survival↗

PubMed2006

A reduced mathematical model of the acute inflammatory response: I. Derivation of model and analysis of anti-inflammation.

The acute inflammatory response, triggered by a variety of biological or physical stresses on an organism, is a delicate system of checks and balances that, although aimed at promoting healing and restoring homeostasis, can result in undesired and occasionally lethal physiological responses. In this work, we derive a reduced conceptual model for the acute inflammatory response to infection, built up from consideration of direct interactions of fundamental effectors. We harness this model to explore the importance of dynamic anti-inflammation in promoting resolution of infection and homeostasis. Further, we offer a clinical correlation between model predictions and potential therapeutic interventions based on modulation of immunity by anti-inflammatory agents.

Acute Disease↗

PubMed2006

3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

3' Untranslated Regions↗

PubMed2005

The contributions of dsRNA structure to Dicer specificity and efficiency.

Dicer processes long double-stranded RNA (dsRNA) and pre-microRNAs to generate the functional intermediates (short interfering RNAs and microRNAs) of the RNA interference pathway. Here we identify features of RNA structure that affect Dicer specificity and efficiency. The data presented show that various attributes of the 3' end structure, including overhang length and sequence composition, play a primary role in determining the position of Dicer cleavage in both dsRNA and unimolecular, short hairpin RNA (shRNA). We also demonstrate that siRNA end structure affects overall silencing functionality. Awareness of these new features of Dicer cleavage specificity as it is related to siRNA functionality provides a more detailed understanding of the RNAi mechanism and can shape the development of hairpins with enhanced functionality.

Base Sequence↗

PubMed2005

Mechanistic insights aid computational short interfering RNA design.

RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.

Algorithms↗

PubMed2004

Rational siRNA design for RNA interference.

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.

Animals↗

PubMed2004

Simultaneous 3-T fMRI and high-density recording of human auditory evoked potentials.

We acquired simultaneous high-field (3 T) functional magnetic resonance imaging (fMRI) and high-density (64- and 128-channel) EEG using a sparse sampling technique to measure auditory cortical activity generated by right ear stimulus presentation. Using dipole source localization, we showed that the anatomical location of the grand mean equivalent dipole of auditory evoked potentials (AEPs) and the center of gravity of fMRI activity were in good agreement in the horizontal plane. However, the grand mean equivalent dipole was located significantly superior in the cortex compared to fMRI activity. Interhemispheric asymmetry was exhibited by fMRI, whereas neither the AEP dipole moments nor the mean global field power (MGFP) of the AEPs showed significant asymmetry. Increasing the number of recording electrodes from 64 to 128 improved the accuracy of the equivalent dipole source localization but decreased the signal-to-noise ratio (SNR) of MR images. This suggests that 64 electrodes may be optimal for use in simultaneous recording of EEG and fMRI.

Acoustic Stimulation↗

PubMed2003

Functional siRNAs and miRNAs exhibit strand bias.

Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statistical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5'-anti-sense (AS) terminal base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal stability (Delta0.5 kcal/mol) at the 5'-AS end than nonfunctional duplexes. Average internal stability of siRNA molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved in duplex unwinding and strand retention by RISC.

Animals↗