siRNAs containing 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2′-F-NMC NTPs

Abstract

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.

siRNA Seed Region Is Divided into Two Functionally Different Domains in RNA Interference in Response to 2′-OMe Modifications

“…A possible explanation is that the functions of nucleotides 6 and 7 may slightly differ according to the siRNA sequence. As for other chemical modifications, it was reported that altritol nucleic acids and 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) modifications at position 7 are also best accommodated and tolerated with the highest performance for the reduction of target gene expression. , …”

RNAs Containing Carbocyclic Ribonucleotides

“…Our laboratory has been interested in studying noncanonical nucleic acid modifications, and we recently reported the synthesis of small interfering RNAs (siRNAs) containing 2′-fluorinated northern -methanocarbacyclic (2′-F-NMC) nucleotides (Figure A). , The 2′-F-NMC scaffold preorganizes the carbocyclic sugar into a pseudo-boat C2′- exo (north) conformation . 2′-F-NMC was generally well tolerated in both the guide and passenger strands of siRNAs with some exceptions, such as positions 1 and 2 on the guide strand. , This prompted us to evaluate the carbocyclic nucleotides, the parent compounds of the NMC scaffold. We have also studied how modifications with low thermal binding affinity such as ( S )-glycol nucleic acid (( S )-GNA), which is also devoid of the 4′-oxygen, mitigates off-target effects of siRNAs …”

Role of a “Magic” Methyl: 2′-Deoxy-2′-α-F-2′-β-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5′-Phosphate Mimics and Mitigation of Off-Target Effects

“…Native NTPs (rCTP, rUTP, dCTP, and dTTP) are efficiently incorporated by the mitochondrial DNA and RNA polymerases POLG and POLRMT, respectively. , Although 2′-F monomers are incorporated at high concentrations by POLRMT, the 2′-F/Me-modified nucleotide analogues are not substrates for either mitochondrial polymerase. − The inability of POLG to incorporate 2′-F/Me-modified residues can be readily explained by an unfavorable interaction between the 2′-Me moiety and the “gatekeeper” residue Tyr-951 at the active site. When a 2′-F/Me CMP from a refined duplex structure (C3′- endo pucker) is superimposed on the incoming dCTP in the crystal structure of the ternary POLG complex (PDB ID 4ZTZ), the gem -hetero-substituted methyl group points directly into the ring of that tyrosine thereby creating a clash (Figure ).…”

“…Native NTPs (rCTP, rUTP, dCTP, and dTTP) are efficiently incorporated by the mitochondrial DNA and RNA polymerases POLG and POLRMT, respectively. 40,75 Although 2′-F monomers are incorporated at high concentrations by POLRMT, the 2′-F/Me-modified nucleotide analogues are not substrates for either mitochondrial polymerase. 15−17 The inability of POLG to incorporate 2′-F/Memodified residues can be readily explained by an unfavorable interaction between the 2′-Me moiety and the "gatekeeper" residue Tyr-951 at the active site.…”