We recently reported the synthesis of 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2′-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2′-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5′ phosphate, suggesting that the 2′-F-NMC is a poor substrate for 5′ kinases. In mice, the 2′-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2′-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5′-phosphate mimic 5′-(E)-vinylphosphonate was attached to the 2′-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2′-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2′-F-NMC. Finally, the 5′-triphosphate of 2′-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.
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Date of publication : 12 February 2021; Nucleic Acids Research, gkab050,
Author Information: Masaaki Akabane-Nakata (1), Namrata D Erande (1), Pawan Kumar (1), Rohan Degaonkar (1), Jason A Gilbert (1), June Qin (1), Martha Mendez (1), Lauren Blair Woods (1), Yongfeng Jiang (1), Maja M Janas (1), Derek K O’Flaherty (1), Ivan Zlatev (1), Mark K Schlegel (1), Shigeo Matsuda (1), Martin Egli(2), Muthiah Manoharan (1)
(1) Alnylam Pharmaceuticals, 675 West Kendall Street, Cambridge, MA 02142, USA
(2) Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA