Nucleic acids for inhibiting expression of pros1 in a cell
a technology of nucleic acids and pros1, which is applied in the field of nucleic acid products, can solve the problems of complex process for detecting potent nucleic acid silencing triggers with minimal off-target effects, severe limitations, and disability at a young ag
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example 1
of Building Blocks
[0567]The synthesis route for DMT-Serinol (GalNAc)-CEP and CPG as described below is outlined in FIG. 1. Starting material DMT-Serinol(H) (1) was made according to literature published methods (Hoevelmann et al. Chem. Sci., 2016, 7, 128-135) from commercially available L-Serine. GalNAc(Ac3)—C4H8—COOH (2) was prepared according to literature published methods (Nair et al. J. Am. Chem. Soc., 2014, 136 (49), pp 16958-1696), starting from commercially available per-acetylated galactose amine. Phosphitylation reagent 2-Cyanoethyl-N,N-diisopropylchlorophosphor-amidite (4) is commercially available. Synthesis of (vp)-mU-phos was performed as described in Prakash, Nucleic Acids Res. 2015, 43(6), 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), 7581-7592. Synthesis of the phosphoramidite derivatives of ST43 (ST43-phos) as well as ST23 (ST23-phos) and similar can be performed as described in WO2017 / 174657.
[0568]DMT-Serinol(GalNAc) (3)
[0569]HBTU (9.16 g, 24.14 mmol) w...
example 2
eotide Synthesis
[0576]Example compounds were synthesised according to methods described below and known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.
[0577]Downstream cleavage, deprotection and purification followed standard procedures that are known in the art.
[0578]Oligonucleotide syntheses was performed on an AKTA oligopilot 10 using commercially available 2′O-Methyl RNA and 2′Fluoro-2′Deoxy RNA base loaded CPG solid support and phosphoramidites (all standard protection, ChemGenes, LinkTech) were used. Synthesis of DMT-(S)-Serinol(GalNAc)-succinyl Icaa CPG (7) and DMT-(S)-Serinol(GalNAc)-CEP (5) are described in example 1.
[0579]Ancillary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile (2O) and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). Coupli...
example 3
rand Formation
[0585]Individual single strands were dissolved in a concentration of 60 OD / ml in H2O. Both individual oligonucleotide solutions were added together in a reaction vessel. For easier reaction monitoring a titration was performed. The first strand was added in 25% excess over the second strand as determined by UV-absorption at 260 nm. The reaction mixture was heated to 80° C. for 5 min and then slowly cooled to RT. Double-strand formation was monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80° C. again and slowly cooled to RT. This procedure was repeated until less than 10% of residual single strand was detected.
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