Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity

Abstract

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.

Graphical Abstract

Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity.

Figure 1.

(A) Structures of canonical phosphodiester backbone and (B) ⁱE-VP backbone.

Scheme 1.

Synthesis of ⁱE-VP-linked dimer phosphoramidites (9a, 9b, and 9c). Reagents and conditions: (i) TBDPSCl, Imidazole/DMF, rt, overnight, 1a: 99%, 1b: 95%; (ii) BzCl, DIPEA/CH₂Cl₂, rt, 4 h, 2a: 78%, 2b: N.D.^a; (iii) 3% trichloroacetic acid, triethylsilane/CH₂Cl_2, rt, 1 h, 3a: 91%, 3b; 63% (2 steps); (iv) IBX/CH₃CN, 85°C, 2 h; (v) CBr₄, PPh₃/CH₂Cl₂, 0°C then rt, 2 h, 5a: 52% in 2 steps, 5b: 56% in 2 steps; (vi) dimethylphosphite, triethylamine/DMF, rt, overnight, 6a-E: 52%, 6b-E: 55%, 6a-Z: 15%, 6b-Z: 22%; (vii) 5′-O-DMTr-uridine-3′-H-phosphonate methyl ester (2′-OMe: S2a or 2′-F: S2b) or 5′-O-DMTr-2-N-(iso-butyryl)- 2′-OMe-guanosine-3′-H-phosphonate methyl ester (S2c), Pd(OAc)₂, dppf, propylene oxide/THF, 70°C or 75°C, 4 h-overnight, 7a: 57%, 7b: 64%, 7c: 30%; (viii) 0.1 M TBAF/THF, rt, 0.5 h, 8a: 94%, 8b: 47%, 8c: 85%; (IX) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA/CH₂Cl₂, 0°C then rt, 0.5 h, 9a: 80%, 9b: 68%, 9c: 57%; ^aNot determined (see materials and methods).

Figure 2.

Impact of incorporating ⁱE-VP into oligonucleotide backbone on nuclease and reverse transcriptase activity. (A) Nuclease digestion test using P-O3′ bond-cleaving 5′-phosphate (P)-dependent 5′-exonuclease (Terminator™, 8.5 mU/μl), P-O5′-cleaving 5′-exonuclease (BSP II, 0.25 U/ml), and P-O3′-bond cleaving snake venom phosphodiesterase I (SVPD, 4 mU/ml). R = PO₃^– for ON1 and 2, R = H for ON3 and 4. (B) Reverse transcription by Superscript II and AMV RT using ⁱE-VP-modified RNA template. *Full-length, extended product. **Truncated product stopped at position of ⁱE-VP (6–7 or 10–11) on templates. ***Minor truncated products stopped at phosphorothioate modification on templates.

Figure 3.

Position-dependent impact of ⁱE-VP backbone on siRNA efficacy and impact of ⁱE-VP backbone on thermal stability of siRNA duplex. Silencing efficacy change was defined by eq 1. Red bar graphs indicate siRNAs having neutral or enhanced silencing efficacy compared to control siRNAs. Black bar graphs indicate ⁱE-VP-modified siRNAs showed lower efficacy than that of control siRNAs. See Table 1 and supporting information for sequences, and Supplementary Table S7 for IC₅₀ values used for the calculation of efficacy changes. *Efficacy change that exceed –300% was set as an artificial difference to -300% in the graph. Comparative T_m of siRNA duplex containing ⁱE-VP and corresponding control siRNA duplex was measured in 10 mM Sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 0.1 mM EDTA. ΔT_m = T_m^VP – T_m^Ctrl. T_m values were determined in triplicate experiments.

Figure 4.

SNPs discrimination properties of ⁱE-VP-modified siRNA. (A) Sequences of siRNAs (^CtrlD15 and ^VPD21), target mRNAs with or without single mismatch (mm) base (target and mm-target, respectively), and IC₅₀ values. (B) Conceptual scheme of ON and OFF-target silencing of ^VPD21. (C) Passive uptake of siRNA and dose response of ^VPD21 and ^CtrlD15 in the presence of target or mm-target mRNAs. (D) Lipid-mediated uptake and dose response of ^VPD21 and ^CtrlD15 in the presence of target or mm-target mRNAs. ^aCtrlD15 and ^VPD21 consist of ^CtrlG15/P15 and ^VPG21/P15, respectively; ^bUppercase and underlined uppercase represent 2′-OMe and 2′-F, respectively. 5′-end phosphate is represented as ‘5′-P’. Inter-nucleotide phosphorothioate and (E)-vinylphosphonate linkages are indicated by ‘#’ and vp, respectively. Passenger strands have 3′-end tetraethylene glycol (Teg)-linked cholesterol (Chol) conjugate. ^cIC₅₀ was calculated based on duplicate experiments. ^dN.D. = Not determined.

Figure 5.

Introduction of ⁱE-VP next to the SNP site enhances discrimination based on a single nucleotide difference in the target site. GalNAc conjugated siRNAs with (^VPD24) and without (^CtrlD18) the introduction of the ⁱE-VP next to the SNP site (^VPD24 and ^CtrlD18, respectively), were injected at 10 mg/kg dose level SC at ∼12 weeks BAC-HD mice. The level of mutant and WT huntingtin proteins was evaluated using western blot (Protein Simple) in mice livers at two weeks post injection. N-5,6, one-way Anova with Bonferroni correction for multiple comparisons. (A) Level of mutant protein expression. Both compounds induce robust silencing. (B) Level of WT protein expression. ^CtrlD18 induces WT protein silencing which is significantly reduced with ^VPD24 compound.