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

Abstract

Abstract:We describe the synthesis, thermal stability, structural and RNase H activation properties of 2′β-fluoro-tricyclo nucleic acids (2'Ftc-ANA). Three 2'F-tc-ANA nucleosides (T, 5Me C and A) were synthesized starting from a previously described fluorinated tricyclo sugar intermediate. NMR analysis and quantum mechanical calculations indicate that 2'F-tc-ANA nucleosides prefer sugar conformations in the East and South regions of the pseudorotational cycle. UV-melting experiments revealed that non-consecutive insertions of 2'F-tc-ANA units in DNA reduce the affinity to DNA and RNA complements. However, an oligonucleotide with 5 contiguous 2'F-tc-ANA-T insertions exhibits increased affinity to complementary RNA. Moreover, a fully modified 10-mer 2′F-tc-ANA oligonucleotide paired to both DNA (+1.6°C/mod) and RNA (+2.5°C/mod) with significantly higher affinity compared to corresponding unmodified DNA, and similar affinity compared to corresponding tc-DNA. In addition, CD spectroscopy and molecular dynamics simulations indicate that the conformation of the 2′F-tc-ANA/RNA duplex is similar to that of a DNA/RNA duplex. Moreover, in some sequence contexts, 2′F-tc-ANA promotes RNase H-mediated cleavage of a complementary RNA strand. Taken together, 2′F-tc-ANA represents a nucleic acid analogue which offers the advantage of high RNA affinity while maintaining the ability to activate RNase H, and can be considered a prospective candidate for gene silencing applications.

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 cells were incubated for 24 ha t3 78Ci na5% CO 2 atmosphere in the RPMI-1640 medium supplemented with 10 %f etal bovine serum (FBS), vitamins, and antibiotics. After incubation, the cells were transfected with different concentrations of gapmers (1,5,20,50,100,200, 500, 1000 nm)b yu sing Lipofectamine 2000 and incubated for 24 h. Next, the MTT assay was performed. The medium was discarded from the cells, then 150 mLo fM TT solution (0.5 mg mL À1 )i nm edium without phenol red were added.…”

“…Previously,i th as been shown that nucleotide conformation and orientation of substituentsp lay an important role for activity.T he incorporation of a3 '-C-hydroxymethyl-arabino nucleotide, with af lexible S-type conformation, or ap iperazino-functionalized-C3'-O2'-linked arabino nucleotide, with ar estricted E-type conformatione nhanced cleavage activity.T he orienta-tion of substituents into the major groove, away from the minor groove were RNase Hi sk nown to bind in aD NA/RNA duplex, was argued as important for tolerability of the modified nucleotides in the initial binding region of RNase H. [1] Combining the 2'-F-ANAm odification with nucleotides that have reduced recognition for RNase Hseems to be asuccessful strategy.H owever,p reviousw ork has established that combining the 2'-F-ANAm odificationw ith the tricyclo-ANAm odification maintainedt he good binding properties and nuclease resistancew hile allowing forRNase Ha ctivation. [5] Selectivity of AON gapmerss hould be of great interest for therapeutic AONs, because off-target effects caused by low specificity potentially lead to toxicity. [10,11] This prompted us to investigate the mismatch discrimination of ON10 with respect to RNase Hc leavage to establish whetheri mproved discrimination towards single-nucleotide polymorphism (SNP) could be observed for ag ap-region-modified antisense gapmer relative to the reference gapmer with an all-DNA gap region.Bymutating one nucleobase at at ime along the 8-mer DNA-based gap region on the complementary RNA, resulting in eight singly mismatched target-RNA strands (T1-T8), the RNase H-induced cleavage patterns were evaluatedr elative to the reference ON8.T he resultsare given in Ta ble2.…”

“…[1] Many chemicalm odifications have been introduced into AONs to address the long-lasting challenges includingb iostability,t argeting specificity,a nd delivery. [2][3][4][5] However,s ome nucleotide modifications that increaset he binding affinityo fA ONs towards RNA targets have been reported to impede the recruitment of RNase H. [1] For instance, incorporation of modified nucleotides with aC 3 '-endo ribose-ring conformation, such as locked nucleic acid (LNA)o rO 2 '-alkylated RNA nucleotides,significantly reduce or halt RNase Hr ecruitment. [1,6,7] This can be circumvented by using so-called antisense gapmers having high-affinity nucleotidest owards the two terminal regions flanking ac entral contiguouss tretch of DNA nucleotides ("the gap"), [8,9] ad esign, which mostf requently has been explored as oligonucleotides containng phosphorothioate linkages throughout (all-PS).…”

“…For AONs, recruitment of RNase H has proved to be a successful strategy to induce cleavage of the target RNA strand of an AON/RNA heteroduplex . Many chemical modifications have been introduced into AONs to address the long‐lasting challenges including biostability, targeting specificity, and delivery . However, some nucleotide modifications that increase the binding affinity of AONs towards RNA targets have been reported to impede the recruitment of RNase H .…”

Applications of Ruthenium Complexes Covalently Linked to Nucleic Acid Derivatives

“…Indeed, in the antisense strategy a chemically modified oligonucleotide (antisense oligonucleotide, ASO) recognises a stretch of mRNA through complementary base-pairing and the resulting steric blocks prevents translation [ 116 ]. Alternatively, ASOs can recruit RNase H which catalyses the cleavage of the RNA part of the ASO-mRNA duplex [ 117 , 118 ]. The development of molecules suited for the antisense strategy and gene silencing could serve as new anticancer agents [ 119 ].…”