title: Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds.
abstract: 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.
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
authors.forename: Shannen
authors.initials: S
authors.lastname: Lau
authors.forename: Bim
authors.initials: B
authors.lastname: Graham
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Boyd
authors.forename: Colin W
authors.initials: CW
authors.lastname: Pouton
authors.forename: Paul J
authors.initials: PJ
authors.lastname: White