commercially supplied amine-modified sirnas may require ultrafiltration prior to conjugation with amine-reactive compounds提供商业amine-modified sirnas之前可能需要超滤与amine-reactive共轭化合物.pdfVIP

SAGE-Hindawi Access to Research Journal of Nucleic Acids Volume 2011, Article ID 154609, 5 pages doi:10.4061/2011/154609 Research Article Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds Shannen Lau, Bim Graham, Ben J. Boyd, Colin W. Pouton, and Paul J. White Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia Correspondence should be addressed to Paul J. White, paul.white@ Received 10 November 2010; Accepted 8 February 2011 Academic Editor: Daisuke Miyoshi Copyright © 2011 Shannen Lau et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 aceta