These experiments to mimic recommended conditions for long oligonucleotide synthesis.6 A 6 min coupling time was used with 0.25M ETT as the activator for all RNA monomers.7 We compared DNA, TBDMS, and TOM monomers as well as the impact of our two universal supports: Universal Support (US) III PS and Glen UnySupportTM CPG.
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Our methods are as follows: Synthesis. On an ABI 394 synthesizer, a 20mer oligonucleotide was synthesized on a 1.0 umol support, with the following sequence using DNA, TBDMS, and TOM phosphoramidites: 5-UUG UUC UUA UUG UUC UUA UU*-3 *For DNA control, T was used in place of U. Cleavage & Deprotection. Each oligonucleotide was deprotected according to support recommendations. For oligonucleotides synthesized using US III PS, oligonucleotides were incubated in 2M ammonia in methanol for 60 min at RT. Without drying down, an equal volume of AMA was added to the solution, and deprotection was continued for 10 min in a 65 water bath. The solution was filtered and dried in a sterile microcentrifuge tube using a steady flow of argon to evaporate the liquid. For oligonucleotides synthesized using Glen UnySupport CPG, oligonucleotides were incubated in AMA for 60 min in a 65 water bath.127-07-1 IUPAC Name The solution was filtered and dried in a sterile microcentrifuge tube using a steady flow of argon to evaporate the liquid. 2-Desilylation. The oligonucleotides were redissolved in DMSO (115 mL) and warmed in a 65 water bath until fully dissolved. TEA (60 mL) and TEAHF (75 mL) were added to the reaction.9005-65-6 SMILES The mixture was heated in a 65 water bath for 2.5 h. The reaction was quenched by adding 750 mL RNA quenching buffer. Desalting. To remove reaction conditions from the previous step, the oligonucleotide was desalted
using a Glen Gel-PakTM 1.0 desalting column. The recommended conditions were followed, and crude oligonucleotides were eluted in 0.1M RNase-free TEAA and analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Coupling efficiency was calculated based on crude oligonucleotide purity. As expected, DNA phosphoramidite coupling performance was the best of the three followed by TOM phosphoramidites and then TBDMS phosphoramidites (Table 1). Crude purity of TOM- and TBDMS-prepared oligonucleotides synthesized on US III PS were 80.PMID:30252267 1 and 77.6%, respectively (Figure 2). Based on MS and RP-HPLC analysis of our crude RNA oligonucleotides, we detected a small degree of DMT loss. The full-length DMT-OFF oligonucleotide co-eluted with failure sequences. The loss of the trityl group likely occurred during the drying step necessary for cleavage and deprotection and the 2-desilylation reaction. This was unavoidable and has implications when it comes to choosing a purification method for your long RNA oligonucleotides. This was a contributing factor to the coupling efficiency differences between DNA and RNA in Table 1.
Figure 2. Crude RP-HPLC chromatograms of 20mer oligonucleotides synthesized on US III PS with DNA (blue), TOM (orange), and TBDMS (green) monomers.
Although only 20mer oligonucleotides were synthesized in this exercise, we can use these values to extrapolate the expected overall purity of a 100mer. We recognize a few caveats with this, namely the fact that failure sequences absorb less at 254nm than the full-length oligonucleotide. It is worth remembering that this extrapolation will predict what the chromatogram looks like, rather than the ultimate yield. Using the following equation, a 100mer made of.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