Limits...
Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.

Petrova NS, Chernikov IV, Meschaninova MI, Dovydenko IS, Venyaminova AG, Zenkova MA, Vlassov VV, Chernolovskaya EL - Nucleic Acids Res. (2011)

Bottom Line: A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs.The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type.Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev ave., 8, Novosibirsk 630090, Russia.

ABSTRACT
The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Show MeSH

Related in: MedlinePlus

Accumulation of the cholesterol-contaning siRNAs in KB-8-5 cells. The carrier-free transfection of non-modified siMDR (A), Ch6-siMDR (B) Ch12-siMDR (C) was analyzed by confocal fluorescent microscopy at 100× magnification after 8 h of incubation. Three-channel (RGB) pictures were obtained using staining by Rhodamine Phalloidin (R), binding to the actin filaments; FITC (G), attached to cholesterol-modified and non-modified siRNAs and DAPI (B), intercalating in DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3299988&req=5

gkr1002-F4: Accumulation of the cholesterol-contaning siRNAs in KB-8-5 cells. The carrier-free transfection of non-modified siMDR (A), Ch6-siMDR (B) Ch12-siMDR (C) was analyzed by confocal fluorescent microscopy at 100× magnification after 8 h of incubation. Three-channel (RGB) pictures were obtained using staining by Rhodamine Phalloidin (R), binding to the actin filaments; FITC (G), attached to cholesterol-modified and non-modified siRNAs and DAPI (B), intercalating in DNA.

Mentions: The localization of FITC-labeled Ch6-siMDR and Ch12-siMDR in KB-8-5 cells was studied by confocal fluorescent microscopy (Figure 4) in order to prove the accumulation of the lipophilic siRNAs inside the cells. Non-modified siRNA did not penetrate into the cells (Figure 4A), whereas cholesterol-containing siRNAs accumulated inside the cells effectively after 8 h of incubation (Figure 4B and C). The green signal corresponding to FITC-labeled siRNAs is located in the cytoplasm of the cells as small discrete granules, and the granules are concentrated around the nuclei. Consequently, cholesterol-conjugated siRNA do accumulate in the cell cytoplasm.Figure 4.


Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.

Petrova NS, Chernikov IV, Meschaninova MI, Dovydenko IS, Venyaminova AG, Zenkova MA, Vlassov VV, Chernolovskaya EL - Nucleic Acids Res. (2011)

Accumulation of the cholesterol-contaning siRNAs in KB-8-5 cells. The carrier-free transfection of non-modified siMDR (A), Ch6-siMDR (B) Ch12-siMDR (C) was analyzed by confocal fluorescent microscopy at 100× magnification after 8 h of incubation. Three-channel (RGB) pictures were obtained using staining by Rhodamine Phalloidin (R), binding to the actin filaments; FITC (G), attached to cholesterol-modified and non-modified siRNAs and DAPI (B), intercalating in DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299988&req=5

gkr1002-F4: Accumulation of the cholesterol-contaning siRNAs in KB-8-5 cells. The carrier-free transfection of non-modified siMDR (A), Ch6-siMDR (B) Ch12-siMDR (C) was analyzed by confocal fluorescent microscopy at 100× magnification after 8 h of incubation. Three-channel (RGB) pictures were obtained using staining by Rhodamine Phalloidin (R), binding to the actin filaments; FITC (G), attached to cholesterol-modified and non-modified siRNAs and DAPI (B), intercalating in DNA.
Mentions: The localization of FITC-labeled Ch6-siMDR and Ch12-siMDR in KB-8-5 cells was studied by confocal fluorescent microscopy (Figure 4) in order to prove the accumulation of the lipophilic siRNAs inside the cells. Non-modified siRNA did not penetrate into the cells (Figure 4A), whereas cholesterol-containing siRNAs accumulated inside the cells effectively after 8 h of incubation (Figure 4B and C). The green signal corresponding to FITC-labeled siRNAs is located in the cytoplasm of the cells as small discrete granules, and the granules are concentrated around the nuclei. Consequently, cholesterol-conjugated siRNA do accumulate in the cell cytoplasm.Figure 4.

Bottom Line: A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs.The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type.Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentiev ave., 8, Novosibirsk 630090, Russia.

ABSTRACT
The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Show MeSH
Related in: MedlinePlus