Fluorinated Nucleotide Modifications Modulate Allele Selectivity of SNP-Targeting Antisense Oligonucleotides

Abstract

Antisense oligonucleotides (ASOs) have the potential to discriminate between subtle RNA mismatches such as SNPs. Certain mismatches, however, allow ASOs to bind at physiological conditions and result in RNA cleavage mediated by RNase H. We showed that replacing DNA nucleotides in the gap region of an ASO with other chemical modification can improve allele selectivity. Herein, we systematically substitute every position in the gap region of an ASO targeting huntingtin gene (HTT) with fluorinated nucleotides. Potency is determined in cell culture against mutant HTT (mtHTT) and wild-type HTT (wtHTT) mRNA and RNase H cleavage intensities, and patterns are investigated. This study profiled five different fluorinated nucleotides and showed them to have predictable, site-specific effects on RNase H cleavage, and the cleavage patterns were rationalized from a published X-ray structure of human RNase H1. The results herein can be used as a guide for future projects where ASO discrimination of SNPs is important.

Gapmer Antisense Oligonucleotides Containing 2′,3′‐Dideoxy‐2′‐fluoro‐3′‐C‐hydroxymethyl‐β‐d‐lyxofuranosyl Nucleotides Display Site‐Specific RNase H Cleavage and Induce Gene Silencing

“…The aqueous phase was then extracted with EtOAc (60 mL) and the combined organic phases were dried (MgSO 4 ), filtered, and evaporated to dryness under reduced pressure to give aw hite solid material, which was vacuum-dried for 2h. R f = 0.59 (5 %M eOH in CH 2 Cl 2 ); HRMS (ESI): m/z calcd for C 15 (12 mL) and dropwise added to as uspension of the Nysted reagent (20 %i nT HF, w/w,5 .38 mL, 3.50 mmol) in anhydrous THF (4.60 mL) at À78 8C. TiCl 4 (1 m,2 .80 mL) was then added dropwise at À78 8Ca nd the resulting mixture was stirred for 2h at À78 8C and subsequently allowed to slowly reach room temperature overnight.…”

“…However,c hangingAto Gi nt he targett og ive the wild-type HTT mRNA completely blocked RNase H1 activity. [15] Gapmer ASOs containing 2'-MOE-RNA with an all-PS backbone constitute the most widely investigated class of nucleic acidt herapeutics in the clinic. [16] LNAa nd constrained ethyl (cEt) have been used insteado fM OE in gapmer PS-AONs to improved uplex stability.…”

“…One study aiming for allele selectivity has shown that fluorinated nucleotides, such as 2′‐fluoro‐hexitol nucleic acid (FHNA), positioned as T6 in an AON gapmer (3‐9‐3 design), induce RNase H cleavage of the complementary mutated huntingting (mtHTT) mRNA. However, changing A to G in the target to give the wild‐type HTT mRNA completely blocked RNase H1 activity . Gapmer ASOs containing 2′‐MOE‐RNA with an all‐PS backbone constitute the most widely investigated class of nucleic acid therapeutics in the clinic .…”

Therapeutic siRNA: state of the art

“…Highly electronegative fluorine makes the modified siRNA readily adopt a C3′-endo conformation, providing considerable benefits in binding affinity, with a ΔT m of~2.5°C per modified nucleotide. 32 Interestingly, 2′-arabino-fluoro (2′-Ara-F), an alternative fluoro substitution form, can also be used to modify nucleic acid therapeutics, e.g., antisense oligonucleotides 45,70 and siRNA. 71 Both 2′-O-benzyl and 2′-O-methyl-4-pyridine (2′-O-CH2Py(4)) were well tolerated when they were incorporated on the guide strand of siRNA in vivo 72 (Fig.…”

2′β‐Fluoro‐Tricyclo Nucleic Acids (2′F‐tc‐ANA): Thermal Duplex Stability, Structural Studies, and RNase H Activation

“…[36] Furthermore, the fully modified 2′F-tc-ANA strand is likely very rigid, and some degree of flexibility is required as the enzyme's active site bends the antisense strand so as to correctly position the cleaved phosphate within the binuclear catalytic site, as concluded from NMR [37] and computational studies. [38] In this context, antisense molecules comprising entirely of F-ANA cannot effectively activate RNase H, but acquiring some flexibility, through flexible linkers, leads to triggering a very potent RNase H response.…”