The mechanism of dephosphorylation. Ion-exchange HPLC analysis of the crude mixtures reveals that the product oligonucleotide was usually present in two forms, the first fully deprotected and the second still containing the 3′-tether awaiting further dephosphorylation. The percentage of tethered product ranged from 15% to 35% of the crude oligonucleotide product. Analysis of the crude products from universal supports 4 and 5 reveal that virtually no tethered oligonucleotide is present in the mixture, demonstrating a different mechanism of dephosphorylation. The yields of all UV260 absorbing material were also determined from the ion-exchange HPLC data, These yields are recorded relative to the highest yield, obtained using universal support 5b. One other commercial support 6, whose structure was not revealed by the manufacturer, was also tested and the percentage of tethered product fell in the same range as supports 1-3, indicating that it is dephosphorylated using a similar mechanism. The results are summarized in Table 1. In the second set of experiments, the universal supports were cleaved and deprotected following the manufacturers’ protocols. All supports performed well with 6
Scheme 4. Cleavage/Dephosphorylation of Oligonucleotides Synthesized on Universal Supports 5a,b
the product oligonucleotide present in the crude mixture at levels ranging from 66% to 87%, with amounts of 3′-tethered product all falling below 1.1082744-20-4 custom synthesis 5%. Again the yields of crude product were recorded relative to the universal support 5b, which generated the highest yield. Finally, overall yields of various DNA oligomers (ranging in length from 20mer to 75mer) obtained from nucleoside bound CPG and Universal Supports 5a,b were basically the same. However, the yields of oligomers, prepared on supports 1-4,6, were always somewhat lower than the amount derived from nucleoside bound CPG and/or universal supports 5a,b. RNA SYNTHESIS ON UNIVERSAL SUPPORTS Universal supports by definition should be appropriate for ALL types of oligonucleotide synthesis. In general, they make a lot of sense for DNA synthesis, but
what is the state of play for RNA synthesis From the results outlined in Tables 1 and 2, it is clear that the type exemplified by universal supports 1-3 is inappropriate for RNA synthesis. The dephosphorylation conditions are simply too aggressive for RNA. However, supports 4, 5 and 6 may be compatible with RNA synthesis. For this study, two strands of siRNA were prepared as shown in Table 3. The RNA monomers were protected with the TOM protecting group6 and the conditions used for RNA deprotection had already been validated to produce biologically active siRNA.57-88-5 medchemexpress All four supports, 4,5a,b,6, gave RNA of purity ranging from 62% to 82%, as determined by ion-exchange HPLC.PMID:30137793 The yield from support 4 was low until more aggressive conditions were used for the cleavage/dephosphorylation step, which led to the lowest percentage of the target oligo in the crude mixture. Supports 5a and 5b gave good yields of crude RNA with reasonable purity of the target oligomer.
COMPARISON OF UNIVERSAL SUPPORTS IN RNA# SYNTHESIS Table 3. RNA oligomers produced following a known, successful protocol Universal Support 5a 5a 6* 6* 5b 5b 4 4 Conditions of cleavage/deprotection (c/d) Percentage of full length oligo with 3′-OH after c/d 82% 79% 66% 51% + 34% of 3’tethered oligomer 76% 75% 62% 82% Relative yield of all UV260 absorbing material after c/d## 100% 88% 54% 66% 100% 84% 61% 19%
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