Synthesis, biophysical properties, and RNase H activity of 6’-difluoro[4.3.0]bicyclo-DNA

Abstract

Here we present the synthesis, the biophysical properties, and the RNase H profile of 6’-difluorinated [4.3.0]bicyclo-DNA (6’-diF-bc4,3-DNA). The difluorinated thymidine phosphoramidite building block was synthesized starting from an already known gem-difluorinated tricyclic glycal. This tricyclic siloxydifluorocyclopropane was converted into the [4.3.0]bicyclic nucleoside via cyclopropane ring-opening through the addition of an electrophilic iodine during the nucleosidation step followed by reduction. The gem-difluorinated bicyclic nucleoside was then converted into the corresponding phosphoramidite building block which was incorporated into oligonucleotides. Thermal denaturation experiments of these oligonucleotides hybridized to complementary DNA or RNA disclosed a significant destabilization of both duplex types (ΔT m/mod = −1.6 to −5.5 °C). However, in the DNA/RNA hybrid the amount of destabilization could be reduced by multiple insertions of the modified unit. In addition, CD spectroscopy of the oligonucleotides hybridized to RNA showed a similar structure than the natural DNA/RNA duplex. Furthermore, since the structural investigation on the nucleoside level by X-ray crystallography and ab initio calculations pointed to a furanose conformation in the southern region, a RNase H cleavage assay was conducted. This experiment revealed that the oligonucleotide containing five modified units was able to elicit the RNase H-mediated cleavage of the complementary RNA strand.

Insertion of a methylene group into the backbone of an antisense oligonucleotide reveals the importance of deoxyribose recognition by RNase H

“…12 In addition, phosphate backbone modifications, such as 5′-alkyl modification, 13 alkylphosphonate linkage, 14 mesylphosphoramidate linkage, 15 and boranophosphate, 16 have been shown to influence the cleavage pattern of RNase H. In addition to phosphate backbone modifications, sugar modifications have also reportedly changed preferences for RNase H-DNA/RNA complex formation. 17–23…”

“…12 In addition, phosphate backbone modifications, such as 5′-alkyl modification, 13 alkylphosphonate linkage, 14 mesylphosphoramidate linkage, 15 and boranophosphate, 16 have been shown to influence the cleavage pattern of RNase H. In addition to phosphate backbone modifications, sugar modifications have also reportedly changed preferences for RNase H-DNA/RNA complex formation. [17][18][19][20][21][22][23] Our question was whether the interaction of phosphate groups or deoxyribose rings is more important in the recognition of DNA/RNA duplex by RNase H. To answer this question, we focused on the effect exerted by a methylene group that is inserted into the phosphate backbone. By inserting a methylene group at the 5′-upside or 3′-downside, the distance between phosphates or deoxyribose rings could be changed in a different pattern (Fig.…”