| 1. |
- Bramsen, Jesper B., et al.
(författare)
-
A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity
- 2009
-
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 37:9, s. 2867-2881
-
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.
|
|
| 2. |
- Srivastava, Puneet, et al.
(författare)
-
Five- and Six-Membered Conformationally Locked 2‘,4‘-Carbocyclic ribo-Thymidines : Synthesis, Structure, and Biochemical Studies
- 2007
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 129:26, s. 8362-8379
-
Tidskriftsartikel (refereegranskat)abstract
- Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range iH-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 °C and 1.5 °C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA (≈85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.
|
|
| 3. |
- Wenska, Małgorzata, et al.
(författare)
-
Synthesis of Conformationally Constrained 2'-N,4'-C-Ethylene-Bridged Adenosine (aza-ENA-A)
- 2007
-
Ingår i: Heterocycles. - : Wiley. - 0385-5414 .- 1881-0942. ; 73:1, s. 303-324
-
Tidskriftsartikel (refereegranskat)abstract
- The synthesis of conformationally constrained 2'-N,4'-C-ethylene-bridged adenosine(aza-ENA-A), in which the pentose-sugar is cis-fused with the piperidino skeleton at C2' and C4' centres of the sugar ring, is reported. The corresponding phosphoramidite building block will be used for incorporation into oligo-DNA and -RNA by solid phase synthesis to examine their nuclease stability as well as their application in blocking the translation of the target RNA using the antisense and siRNA approach. 2-Aza-6-oxabicyclo[3.2.1]octane skeleton is assembled through multi-step synthetic manipulation of appropriately protected D-arabinose based sugar precursor. The conversion of appropriate arabino precursor to ribo counterpart was achieved by direct nucleophilic displacement of "ara" positioned 2-(trifluoromethanesulfonyloxy) group in the sugar precursor 8. A high regio- and enhanced stereoselectivity with preferential formation of beta anomer in glycosylation reaction was achieved using Vorbruggen conditions in the absence of any 2-participating group. Coupling step was performed using 1-O-acetyl-3,5-di-O-benzyl-(2-deoxy-2-azido)-4-C-(p-toluoyloxyetliyl)-D- ribofur anose (10) as a glycosyl donor and persilylated N-6-benzoyladenine. Finally, the ring-closure giving the North-type conformationally constrained cis-fused bicyclic aza-ENA-A have been confirmed unambiguously by the long range H-1-C-13 NMR correlation (HMBC), TOCSY, COSY and nOe experiments.
|
|
