Introduction of nucleosides into GNA/RNA improves siRNA drug activity in Nucleic Acid Research

Off target effect is an important factor in the toxic side effects of nucleic acid drugs, and by introducing GNA into the seed region of siRNA drug guide chains, the stability of base pairing between individual nucleotides can be reduced, thereby alleviating off target effects. However, the formation of GNA/RNA is a double stranded structure, and the bases of GNA generally adopt a rotating conformational orientation, which affects the complementary pairing between bases. Researchers from Alnylam Company introduced isoG and isoC nucleosides into GNA to overcome the impact of the rotation orientation change of GNA bases on the activity of siRNA drugs, while reducing the off target effect of siRNA drugs. The article was published in Nucleic Acids Research.

GNA， The structure is shown in the following figure, using a non cyclic ribose structure with only one chiral center in a three carbon main chain structure. It is interesting that the homologous double stranded structure of GNA has better thermal stability than the double stranded structure of DNA and RNA. However, in non homologous heterozygous double stranded DNA/RNA, an increase in G: C content reduces the thermal stability of the double stranded DNA/RNA, which is different from conventional DNA/RNA. The main reason is that in the GNA strand, the rotation orientation of the base conformation (180 °, trans syn) changes the formation of hydrogen bonds, which in turn affects the Watson Crick complementary pairing between bases. At the same time, G: C base pairing also shifts from the large groove to the small groove.

Since in the double stranded structure, the bases of GNA adopt a rotating conformation, it can be imagined that if isoG and isoC are used to replace the G and C bases in GNA, it can change the anti Waston Crick pairing of the bases in GNA, thereby altering the stability of GNA base pairing. The researchers first synthesized two phosphoramidite monomers, GNA iC and GNA iG, and then introduced them into siRNA nucleic acid chains. As expected, the use of isoC and isoG can indeed improve the thermal stability of GNA/RNA double strands.

Subsequently, the researchers compared the effects of introducing nucleosides on activity, and the authors designed GalNAc siRNA drugs targeting hao1 (D1-D3) and Ttr genes (D4-D6) respectively. Among them, D1 and D4 are RNA double stranded, D2 and D5 are GNA-C/G, and D3 and D6 are GNA isoG/isoC. The silencing activity of the target gene in D2 using GNA-C is slightly reduced compared to D1, but the activity in D3 using GNA isoC is equivalent to D1. Compared to D4, the activity of D5 using GNA-G did not change significantly, but the silencing activity of D6 using GNA-isoG was somewhat enhanced.

Next, the author compared the effects of introducing GNA isoG/isoC on off target effects. At four concentrations of 0.1, 1, 10, and 50 nM, there was no significant change in the targeting activity of D2 and D5 using GNAC/G, but the off target effect was significantly improved with increasing drug concentration. The performance of D3 using GNA isoC is similar to D2. However, D6 using GNA isoG showed an increase in CDF migration at high concentrations compared to D5 using GNA-G, but still showed some relief compared to D4.

The in vivo activity of mice showed that the silencing activity of target genes using GNA-C/G was lower than that of RNA double stranded parents, while the silencing activity of target genes using GNA isoG/isoC nucleic acid double stranded parents was better than that of GNA-C/G, but there was still a certain decrease in activity compared to RNA double stranded parents. Through the detection of the guiding chain in the liver, it was found that there is a certain correlation between its activity and metabolic activity. The tolerance of GNA-C/G modification in vivo is lower than that of GNA isoG/isoC.

According to previous reports, the use of GNA can reduce the off target effect of siRNA drugs, but it also affects the in vivo activity of such drugs. The introduction of GNA isoG/isoC nucleosides can reverse the conformational orientation changes of bases, improve the activity of GNA siRNA drugs, and also have some improvement on off target effect. The introduction of GNA can destabilize the siRNA seed region, allowing for the optimization of siRNA drugs by finely tuning individual nucleotides.