Rajendra K. Pandey

Various chemical modifications are currently being evaluated for improving the efficacy of short interfering RNA (siRNA) duplexes as antisense agents for gene silencing in vivo. Among the 2'-ribose modifications assessed to date, 2'deoxy-2'-fluoro-RNA (2'-F-RNA) has unique properties for RNA interference (RNAi) applications. Thus, 2'-F-modified nucleotides are well tolerated in the guide (antisense) and passenger (sense) siRNA strands and the corresponding duplexes lack immunostimulatory effects, enhance nuclease resistance and display improved efficacy in vitro and in vivo compared with unmodified siRNAs. To identify potential origins of the distinct behaviors of RNA and 2'-F-RNA we carried out thermodynamic and X-ray crystallographic analyses of fully and partially 2'-F-modified RNAs. Surprisingly, we found that the increased pairing affinity of 2'-F-RNA relative to RNA is not, as commonly assumed, the result of a favorable entropic contribution ('conformational preorganization'), but instead primarily based on enthalpy. Crystal structures at high resolution and osmotic stress demonstrate that the 2'-F-RNA duplex is less hydrated than the RNA duplex. The enthalpy-driven, higher stability of the former hints at the possibility that the 2'-substituent, in addition to its important function in sculpting RNA conformation, plays an underappreciated role in modulating Watson-Crick base pairing strength and potentially π-π stacking interactions.

With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2′-deoxy-2′-fluoro (2′-F) modification is unique. Furthermore, the 2′-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND: Overexpression of alpha-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD. RESULTS: We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion. CONCLUSION: We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for alpha-synucleinopathies resulting from SNCA overexpression.