Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds
Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modif...
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| Format: | Article |
| Language: | English |
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Wiley
2011-01-01
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| Series: | Journal of Nucleic Acids |
| Online Access: | http://dx.doi.org/10.4061/2011/154609 |
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| author | Shannen Lau Bim Graham Ben J. Boyd Colin W. Pouton Paul J. White |
| author_facet | Shannen Lau Bim Graham Ben J. Boyd Colin W. Pouton Paul J. White |
| author_sort | Shannen Lau |
| collection | DOAJ |
| description | Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions. |
| format | Article |
| id | doaj-art-4a5a87aacb54439aaf6e750a4cbcafbe |
| institution | Kabale University |
| issn | 2090-021X |
| language | English |
| publishDate | 2011-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Nucleic Acids |
| spelling | doaj-art-4a5a87aacb54439aaf6e750a4cbcafbe2025-02-03T06:07:02ZengWileyJournal of Nucleic Acids2090-021X2011-01-01201110.4061/2011/154609154609Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive CompoundsShannen Lau0Bim Graham1Ben J. Boyd2Colin W. Pouton3Paul J. White4Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, AustraliaMonash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, AustraliaMonash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, AustraliaMonash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, AustraliaMonash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, AustraliaConjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions.http://dx.doi.org/10.4061/2011/154609 |
| spellingShingle | Shannen Lau Bim Graham Ben J. Boyd Colin W. Pouton Paul J. White Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds Journal of Nucleic Acids |
| title | Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds |
| title_full | Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds |
| title_fullStr | Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds |
| title_full_unstemmed | Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds |
| title_short | Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds |
| title_sort | commercially supplied amine modified sirnas may require ultrafiltration prior to conjugation with amine reactive compounds |
| url | http://dx.doi.org/10.4061/2011/154609 |
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