Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds
Joint Authors
White, Paul J.
Lau, Shannen
Graham, Bim
Boyd, Ben J.
Pouton, Colin W.
Source
Issue
Vol. 2011, Issue 2011 (31 Dec. 2011), pp.1-5, 5 p.
Publisher
Hindawi Publishing Corporation
Publication Date
2011-05-29
Country of Publication
Egypt
No. of Pages
5
Main Subjects
Abstract EN
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.
American Psychological Association (APA)
Lau, Shannen& Graham, Bim& Boyd, Ben J.& Pouton, Colin W.& White, Paul J.. 2011. Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds. Journal of Nucleic Acids،Vol. 2011, no. 2011, pp.1-5.
https://search.emarefa.net/detail/BIM-450168
Modern Language Association (MLA)
Lau, Shannen…[et al.]. Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds. Journal of Nucleic Acids No. 2011 (2011), pp.1-5.
https://search.emarefa.net/detail/BIM-450168
American Medical Association (AMA)
Lau, Shannen& Graham, Bim& Boyd, Ben J.& Pouton, Colin W.& White, Paul J.. Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds. Journal of Nucleic Acids. 2011. Vol. 2011, no. 2011, pp.1-5.
https://search.emarefa.net/detail/BIM-450168
Data Type
Journal Articles
Language
English
Notes
Includes bibliographical references
Record ID
BIM-450168