standard phosphoramidite chemistry. The addition of multiple palmitates
Tides (ASOs) using
standard phosphoramidite chemistry. The addition of multiple palmitates increased the lipophilicity of the ASOs, which in turn enhanced their cellular uptake compared to ASOs without palmitate. The position of palmitate insertion within the ASO sequence also affected cellular uptake. Specifically, inserting two palmitates at the 5-end of the ASO improved cellular uptake while placing one palmitate at the 5-end and another at the 3-end resulted in reduced cellular uptake compared to ASOs without fatty acids.4
Use of Palmitate Serinol Phosphoramidite
The Palmitate Serinol Phosphoramidite has an advantage over the Palmitate Phosphoramidite in that it dissolves fully in acetonitrile, whereas the latter requires a mixture of acetonitrile and dichloromethane (1:3). Like the Palmitate Phosphoramidite, a 6-minute coupling time is recommended for the serinol version. No additional changes are necessary beyond the standard deprotection and cleavage methods required for the nucleobases.
Preventing Detritylation During RNA Deprotection
In our last issue, we reported on coupling efficiency using various monomers (DNA vs. TOM vs. TBDMS).1 As expected, TOM performed better than TBDMS, which has a more significant impact for longer oligonucleotides. During our experiments, we observed some unavoidable loss of the DMT group while drying the oligonucleotide down between global deprotection/ cleavage and 2-desilylation. This is not ideal, especially for those relying on DMT-ON purification for their RNAs. To follow up on this, we sought a method to minimize detritylation during the workup of RNA. It turns out, we didn’t have to look too far. About 15 years ago, we developed a method to prevent detritylation caused by drying down and tested this on trityl-protected amino-modifiers.2 In DNA oligonucleotides, a nonvolatile base, such as TRIS, is added prior to drying down to prevent detritylation. However, the presence of TRIS inhibits the subsequent 2-desilylation reaction with TEAHF for RNA. The obvious next step was to test this on our RNA sequence: 5-UUG UUC UUA UUG UUC UUA UU-3 After synthesis on our ABI394 with TBDMS phosphoramidites, we used the following procedure: Deprotect oligonucleotide as necessary according to nucleobase protecting groups and support. Add 45 mg Tris base/mL.
TECHNICAL NOTE
Desalt on Glen-PakTM DNA Cartridge. Condition cartridge with 0.39011-90-0 custom synthesis 5 mL ACN, followed by 1 mL 2M TEAA. Load oligonucleotide drop-wise onto Glen-Pak DNA Cartridge. Rinse with 2 mL RNase-free water. Rinse twice with 2 mL 0.5M aqueous sodium hydroxide.179463-17-3 MedChemExpress * Rinse with 2 mL RNAse-free water.PMID:28723023 Elute with 1 mL 75% ACN/RNase-free water. Dry down. 2-Desilylation. Dissolve in DMSO (115 L) and warm in a 65 water bath until fully dissolved. Add TEA (60 L) and TEAHF (75 L). Heat in a 65 water bath for 2.5 h. Quench with RNA quenching buffer (750 L). Desalt on Glen Gel-PakTM following recommended conditions and elute crude oligonucleotide in 0.1M RNase-free TEAA. The resulting oligonucleotide was analyzed by reversephase high-performance liquid chromatography (RP-HPLC).
* In general, NaOH treatment is rather harsh for RNA oligonucleotides and we don’t typically see recommendations like this. Due to the short contact time while on the column, the sodium hydroxide did not negatively impact the RNA integrity, confirmed by the chromatograms. However, it is worth pointing out that this step should be done quickly. While we did not test other possibilities, alternative sodi.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
