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.
Highlights
A series of obstacles impede the successful delivery of short interfering RNA to the cytosol of target cells [1, 2]
Previous studies have shown that a number of thiol-reactive drugs rapidly and selectively bind to endogenous albumin within a few minutes of administration, due to that fact that free thiol groups are not found on the majority of circulating serum proteins except for albumin [9]
Initial attempts to couple the short interfering RNA (siRNA) to bovine serum albumin (BSA) via the use of succinimidyl 4[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), a heterobifunctional cross-linker routinely employed to link amine- to thiol-bearing molecules, met with very limited success
Summary
A series of obstacles impede the successful delivery of short interfering RNA (siRNA) to the cytosol of target cells [1, 2]. Strategies involving conjugation of siRNA to a wide variety of molecules have been employed to overcome these obstacles. These approaches employ amine- [3] or thiol-modified [4, 5] siRNA at 3 or 5 ends of one strand of the siRNA duplex (see [6] for review of conjugation chemistry). The strategy employed involved the use of the common heterobifunctional linker succinimidyl 4[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). Using this approach, the coupling of SMCC to siRNA containing an aminohexyl pendant at the 3 end of the sense strand results in an “activated” siRNA bearing a thiol-reactive maleimide group. The chemistry used to conjugate the siRNA to albumin is not novel; others have conjugated nanoparticles to siRNA using a similar cross-linker, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) [10]
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