| 1. |
- Bramsen, Jesper B., et al.
(författare)
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A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity
- 2009
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 37:9, s. 2867-2881
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Tidskriftsartikel (refereegranskat)abstract
- The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.
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| 2. |
- Bramsen, Jesper B., et al.
(författare)
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A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects
- 2010
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:17, s. 5761-5773
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Tidskriftsartikel (refereegranskat)abstract
- Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5'-end) and complementary sequences in the 3'UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA-target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.
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| 3. |
- Semenyuk, Andrey, 1978-
(författare)
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Novel Methods for Synthesis of High Quality Oligonucleotides
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC.In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties.The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.
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