Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Small interfering RNA (siRNA) is a powerful and highly effective method to regulate gene expression in vitro and in vivo. However, the susceptibility to serum nuclease-catalyzed degradation is a major challenge and it remains unclear whether the strategies developed to improve the stability of siRNA free in serum solution are ideal for siRNA conjugated to nanoparticle surfaces. Herein, we use spherical nucleic acid nanoparticle conjugates, consisting of gold nanoparticles (AuNPs) with siRNA chemisorbed to the surface, as a platform to study how a model siRNA targeting androgen receptor degrades in serum (SNA-siRNA(AR)). In solutions of 10% (vol/vol) FBS, we find rapid endonuclease hydrolysis at specific sites near the AuNP-facing terminus of siRNA(AR), which were different from those of siRNA(AR) free in solution. These data indicate that the chemical environment of siRNA on a nanoparticle surface can alter the recognition of siRNA by serum nucleases and change the inherent stability of the nucleic acid. Finally, we demonstrate that incorporation of 2'-O-methyl RNA nucleotides at sites of nuclease hydrolysis on SNA-siRNA(AR) results in a 10-fold increase in siRNA lifetime. These data suggest that strategies for enhancing the serum stability of siRNA immobilized to nanoparticles must be developed from a dedicated analysis of the siRNA-nanoparticle conjugate, rather than a reliance on strategies developed for siRNA free in solution. We believe these findings are important for fundamentally understanding interactions between biological media and oligonucleotides conjugated to nanoparticles for the development of gene regulatory and therapeutic agents in a variety of disease models.