Gas-Phase Cleavage and Dephosphorylation of Universal Linker-Bound Oligodeoxynucleotides

Abstract

While base-specific support is commonly used for single-column oligodeoxynucleotide synthesis, the universal linker is critical for high-throughput synthesis of potentially thousands of samples in a single run. Here, we report conditions for cleavage and complete dephosphorylation of two commercial universal linkers, UnySupport and UnyLinker, processed in the gas phase (NH(3)) using our custom device. First, we compared the average yield of T10mers over time (15, 30, 60, 120, and 240 minutes, 40 psi, 80°C and 90°C). For samples processed with water added prior to incubation, we discovered a substantial increase in yield compared to those left dry (up to 55%). This was also the case for samples subjected to increases in chamber pressure (10, 20, 30 and 40 psi, 120 minutes, 80°C and 90°C). Next, we compared the effects of increased temperature, pressure and incubation times on the rates of dephosphorylation. We found the optimum conditions to be either 10 psi, 120 minutes at 80°C or 60 minutes at 90°C; in both cases, water added to columns prior to incubation had a substantial effect on rate of reaction as well as overall yield compared with those left dry. Finally, performance between the two linkers was similar enough to conclude each fulfills the desired requirements for mainstream, high-throughput oligodeoxynucleotide cleavage/deprotection and dephosphorylation in the gas phase.

Solid‐Phase Supports for Oligonucleotide Synthesis

“…For deprotection of oligoribonucleotides and oligonucleotides containing 2 -F-derivatized nucleoside residues, AH/EtOH (3:1 v/v) for 6 hr at 55 • C was recommended (Prakash et al, 2005). Deprotection of oligonucleotides assembled on 23b and 23e using gaseous ammonia as a deprotection agent was studied in detail (Jensen et al, 2010). For samples wetted with water prior to the treatment with ammonia, the optimal conditions for the 3 -dephosphorylation were 80 • C for 120 min or 90 • C for 60 min at an ammonia gas pressure of 10 psi (0.69 Bar).…”

Expansion of Phosphoramidite Chemistry in Solid-Phase Oligonucleotide Synthesis: Rapid 3′-Dephosphorylation and Strand Cleavage

“…Because a nucleophilic attack on the resulting phosphodiester is difficult to achieve, the subsequent cyclization and release of ONs (dephosphorylation at the 3′-terminus) are considered as the rate-limiting step. Eventually, ON binds to the universal linker at the 3′-terminus phosphate (ON adduct) remains as a side product under insufficient cleavage conditions such as nonheating alkaline conditions. , Although universal linkers have been applied in manufacturing-scale synthesis and high-throughput ON synthesis, , the milder method for removing the linkers will lead to more practical ON synthesis using phosphoramidite chemistry. In addition, acyclic universal linkers, , which are strongly bound to a solid support via amide or urea bonds, have also been reported.…”

Oligodeoxynucleotide synthesis using protecting groups and a linker cleavable under non-nucleophilic conditions

“…One methodology is to use nucleosidic phosphoramidites without base protection. [4][5] Sekine and his coworkers have proposed a method using activated phosphite ( Figure 5.3); where they activated phosphoramidite 5.1 with 1hydroxybenzotriazole (HOBt). 5b The derived triester 5.3 can be highly selective with the 5ʹ hydroxyl group of 5.2.…”

“…However, functionalities in organic chemistry suitable for the need are limited. The ones used in the literatures were the palladiumremovable allyl groups 4 and the photo-labile o-nitrobenzyl linker 5 . ODN synthesis methods relying on them suffer from drawbacks such as high cost of palladium, difficulty to remove transition metal, and DNA photo damages.…”

“…The combined aqueous solution was desalted over dextran desalting column (5K MWCO, 10 mL) with DI-H2O as the eluent. Fractions were collected every milliliter.Fractions 3, 4,5,6 were combined and concentrated. The obtained ODN was dissolved in 50 µL H2O, and 20 µL solution was injected into RP HPLC.…”