Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo.

Torgeir Holen; Svein Erik Moe; Jan Gunnar Sørbø; Trine J Meza; Ole Petter Ottersen; Arne Klungland

doi:10.1093/nar/gki785

Home/Archive/PubMed

PubMed · 16113241

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo.

Torgeir Holen·Svein Erik Moe·Jan Gunnar Sørbø·Trine J Meza·Ole Petter Ottersen·Arne Klungland

View original source ↗Export metadata ↓

Abstract

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5' and 3'ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5' terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions.

Explore related subjects

Aquaporin 4 Aquaporins Base Pairing Blotting, Western Flap Endonucleases HeLa Cells Humans Mutation RNA Interference RNA, Messenger RNA, Small Interfering Journal Article Research Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Torgeir Holen, Svein Erik Moe, Jan Gunnar Sørbø, Trine J Meza, Ole Petter Ottersen, Arne Klungland. 2005-08-19. Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo.. https://doi.org/10.1093/nar%2Fgki785

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Neuromyelitis optica and anti-aquaporin-4 antibodies: widening the clinical phenotype.

Explore the source record for details and available documents.

Aquaporin 4↗

PubMed2006

Astrocyte end-feet in germinal matrix, cerebral cortex, and white matter in developing infants.

Astrocyte end-feet ensheathe blood vessels in the brain and are believed to provide structural integrity to the cerebral vasculature. We sought to determine in developing infants whether the coverage of blood vessels by astrocyte end-feet is decreased in germinal matrix (GM) compared with cerebral cortex and white matter (WM), which may cause fragility of the GM vasculature. Therefore, we evaluated the perivascular coverage by astrocyte end-feet in these areas. We double-labeled the brain sections with astroglial markers [glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), and S-100beta] and a vascular marker, laminin. Perivascular coverage by GFAP+ astrocyte end-feet increased consistently as a function of gestational age (GA) in cortex and WM from 19 to 40 wk. Compared with GFAP, AQP4+ astrocyte end-feet developed at an earlier GA, ensheathing about 63% of blood vessels for 23-40 wk in cortex, WM, and GM. Coverage by GFAP+ perivascular end-feet was decreased in GM compared with cortex and WM from 23 to 34 wk. There was no difference in the coverage by AQP4+ end-feet among the three areas in these infants. The expression of AQP4, a water channel molecule, in the astrocyte end-feet was not significantly different between premature and mature infants, suggesting similar risk of brain edema in preterm and term infants in pathologic conditions. More importantly, the lesser degree of GFAP expression in astrocyte end-feet of GM compared with cortex and WM may reflect a cytoskeletal structural difference that contributes to the fragility of GM vasculature and propensity to hemorrhage.

Aquaporin 4↗

PubMed2005

Redistribution of the water channel protein aquaporin-4 and the K+ channel protein Kir4.1 differs in low- and high-grade human brain tumors.

The blood-brain barrier (BBB) regulation is characterized by an interplay between endothelial cells, subendothelial basal laminae and astrocytic cells. Astroglial cells are highly polarized by the differentiation of perivascular membrane domains. These domains are characterized by the aggregation of, among other molecules, the water channel protein aquaporin-4 (AQP4), the dystrophin-dystroglycan complex, and the inwardly rectifying potassium channel protein Kir4.1. Normally, this ion channel plays an important role in spatial buffering of extracellular K(+) in the central nervous system, which only can be performed due to the non-uniform distribution of Kir4.1 across the surface of the glial cell. In this study, we observed a mislocalization of Kir4.1 in various human brain tumors (low- and high-grade astrocytomas and oligodendrogliomas), suggesting that buffering capacity of glial cells may be compromised, leading to water influx (cytotoxic edema). Interestingly, whereas dystrophin remained regularly restricted at the endfeet membranes in all cases investigated, AQP4 was found to be redistributed only in high-grade astrocytomas, not in low-grade astrocytomas. If the mechanisms of redistribution of AQP4 and Kir4.1 are different in low- and high-grade gliomas, this may suggest that the mechanisms of clustering of AQP4 and Kir4.1 at the glial endfeet membrane domains are also different. The redistribution of AQP4 in glioblastoma cells is discussed as a reaction to the vasogenic edema, as induced by the breakdown of the BBB, to facilitate reabsorption of excess fluid.

Aquaporin 4↗