Minor-Groove-Modulating Adenosine Replacements Control Protein Binding and RNAi Activity in siRNAs

Abstract

Short-interfering RNAs (siRNAs) are common tools in molecular biology, however the development of RNAi-based therapeutics is limited by immunostimulatory and non-specific effects mediated by off-target RNA-binding proteins. PKR and ADAR1 are two proteins implicated in RNAi off-target effects, and share a common means of interaction with siRNAs through double-stranded RNA binding motifs (dsRBMs). Here we report the site-specific introduction of N2- propargyl 2-aminopurine into siRNAs and subsequent conversion to two bulky products via copper-catalyzed azide alkyne cycloaddition (CuAAC) with either Nazidoacetyl-mannosamine azide or N-ethylpiperidine azide. We observed position-specific effects on RNAi activity for modifications made to both the passenger and guide strands. These findings are rationalized in light of recent structural studies of components of the RNA-induced silencing complex (RISC) and RISC-loading complex (RLC). The most active siRNAs were assayed for binding affinity to the RNA-dependent protein kinase (PKR) and adenosine deaminase that acts on RNA 1 (ADAR1). PKR binding was significantly reduced by multiple modifications, regardless of size, and ADAR1 binding was reduced in a position and size-sensitive manner. Our findings present a strategy for designing siRNAs that reduce off-target dsRBM-protein binding while retaining native RNAi activity.

Base Modification Strategies to Modulate Immune Stimulation by an siRNA

“…Previous RNAi studies with a different sequence had shown that position 2 of the guide strand is sensitive to this modification whereas modification at position 14 was tolerated. [25] Here, all eight positions tested showed significantly reduced RNAi activity, with position 20 as the most tolerant to modification (~ two-fold reduction in knockdown compared to the unmodified siRNA). On the other hand, modification on the Hoogsteen face at positions 12, 15, and 20 in this sequence retains RNAi activity (Figure 4).…”

“…However, base modifications to an siRNA guide strand pose the risk of disrupting essential hydrogen bonding crucial for the formation of the A-form duplex needed to activate RNAi. Previously, our lab has developed replacements for adenosine in siRNAs that maintain base pairing with uridine while altering either the Hoogsteen (7-triazolo-8-aza-7-deazaadenosines) [22–23] or WC face ( N 2 -alkyl-2-aminopurines) [24–25] (Scheme 1). …”

“…[22, 24] This particular azide was chosen because the resulting triazoles are sterically demanding, but are only minimally destabilizing to RNA duplexes. [22, 25] To test the immunostimulatory properties of the modified siRNAs, we transfected the duplex oligonucleotides into human PBMCs and quantified TNFa production after 16–20 h of incubation (Figure 2B). We found that, for this sequence, modifying positions near the 5′ end of the siRNA ( i.e.…”

Novel Modifications in RNA

“…These include fluorescent groups for detection or imaging and groups that alter tissue delivery and cellular uptake of the RNA (9, 23). Novel reactive “handles” also allow one to ligate fragments together generating large functional RNAs from smaller synthetic strands or to diversify an RNA structure from a single common intermediate for structure/activity relationship studies (24–26). Early work on this topic included methods to introduce aliphatic amines, thiols, and aldehydes into RNA (27–28).…”

“…Furthermore, triazole formation with the alkyne-bearing RNA strands was efficient with primary azides, copper sulfate, sodium ascorbate and the copper binding ligand tris-(hydroxypropyltriazolylmethyl)amine. Modification of siRNAs with this procedure allowed us to probe the effect of varying minor groove substituents on RNA duplex stability, base pairing specificity, RNA interference, and the binding of known siRNA-binding proteins (26, 30). For instance, the N-ethylpiperidine derivative (9, Figure 3) had miminal effect on RNAi activity at multiple positions in an siRNA but substantially reduced off-pathway protein binding (26).…”