Modulation of thermal stability can enhance the potency of siRNA

Abstract

During RNA-induced silencing complex (RISC) assembly the guide (or antisense) strand has to separate from its complementary passenger (or sense) strand to generate the active RISC complex. Although this process was found to be facilitated through sense strand cleavage, there is evidence for an alternate mechanism, in which the strands are dissociated without prior cleavage. Here we show that the potency of siRNA can be improved by modulating the internal thermodynamic stability profile with chemical modifications. Using a model siRNA targeting the firefly luciferase gene with subnanomolar IC50, we found that placement of thermally destabilizing modifications, such as non-canonical bases like 2,4-difluorotoluene or single base pair mismatches in the central region of the sense strand (9-12 nt), significantly improve the potency. For this particular siRNA, the strongest correlation between the decrease in thermal stability and the increase in potency was found at position 10. Controls with stabilized sugar-phosphate backbone indicate that enzymatic cleavage of the sense strand prior to strand dissociation is not required for silencing activity. Similar potency-enhancing effects were observed as this approach was applied to other functional siRNAs targeting a different site on the firefly luciferase transcript or endogenously expressed PTEN.

Scheme 1.

Synthesis of 5-nitroindole ribonucleoside phosphoramidite. (i) KOH, 2,4-dichlorobenzyl chloride, 18-crown-6/THF, room temperature, overnight, 92%; (ii) HOAc-HBr, 0–25°C, 3 h; (iii) NaH, 5-nitroindole/MeCN, ice bath to room temperature, 4 h, Ar atm. 60%; (iv-a) BCl₃/CH₂Cl₂, −78°C for 2 h, −40°C for 2 h, α/β ratio 1:1; 68% and (iv-b). DMT-Cl, DMAP/Py, overnight, Ar. atm., combined yield: 64% (β isomer 5a (33%), α isomer 5b (11%) and mixture 21%); (v) TBDMS-Cl, AgNO₃, Py/THF, room temperature, overnight, combined yield: 75% (6a: 35%, 6b: 23% and mixture: 17%); (vi) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIEA/CH₂Cl₂, room temperature, overnight, 83%.

Figure 1.

Effect of base pair mismatches across the sense strand sequence of siRNA-1 on potency relative to the parent duplex plotted as IC₅₀ (parent)/IC₅₀ (modified).

Figure 2.

Base modifications in the central region of the sense strand of siRNA-1: the positions of the parent duplex, which were replaced against the nucleotide analogs, are shown in bold. (a) 2,4-difluorotoluene (F and dF); (b) 5-nitroindole (N and dN); (c) inosine (I and dI); (d) nebularine (Neb and dNeb); (e) 2-aminopurine (2AP and d2AP).

Figure 3.

Effect of nucleobase modifications in positions 9–12 of the sense strand of siRNA-1: (A) ribo- and deoxyribo versions of non-polar nucleobase isosteres 2,4 difluorotoluene (F, dF) and 5-nitroindole (N, dN); (B) ribo- and deoxyribo versions of the nucleobase analogs 2-aminopurine (2AP, d2AP), inosine (I, dI) and nebularine (Neb, dNeb); changes in potency relative to the parent siRNA are expressed as IC₅₀ (parent)/IC₅₀ (modified).

Figure 4.

Correlation between the decrease in IC₅₀ and the thermal destabilization of the modified siRNAs compared to the parent unmodified siRNA-1 calculated as ΔT_m = T_m(parent)−T_m(modified). Modifications were incorporated in (A) position 9, (B) position 10, (C) position 11 and (D) position 12.

Figure 5.

Head-to-head comparison of a selected set of thermally destabilizing modifications in the sense strand of siRNA-1: (A) representative example of dose-dependent silencing of firefly luciferase as percent untreated control; (B) potency enhancement of the modified siRNAs relative to the parent duplex (average of three independent transfection experiments).

Figure 6.

(A) Effect of abasic modifications in positions 9–12 of the sense strand of siRNA-1; (B) effect of 2′-O-methyl and/or phosphorothioate modifications between positions 9 and 10 of the sense strands of siRNA-1 and siRNA-2; changes in potency relative to the parent siRNA are expressed as IC₅₀ (parent)/IC₅₀ (modified); P = S, phosphorothioate linkage between positions 9 and 10; g, 2′-O-methylguanosine.

Figure 7.

Effect of base pair mismatches in positions 9,10 and 2,4-difluorotoluene (F) in positions 9–12 of the sense strand of siRNA-4 targeting PTEN; changes in potency relative to the parent siRNA are expressed as IC₅₀ (parent)/IC₅₀ (modified).