A High‐Throughput Process for the Solid‐Phase Purification of Synthetic DNA Sequences

Abstract

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences.

An Alternate Process for the Solid‐Phase Synthesis and Solid‐Phase Purification of Synthetic Nucleic Acid Sequences

“…The detailed protocols reported herein undeniably represent an improvement over a previously reported process (Grajkowski et al, 2016(Grajkowski et al, , 2017 for solid-phase purification of synthetic DNA sequences based on a simpler chemistry and the use of proficient phosphoramidites (i.e., 5, 6, and 9) for incorporating strategic elements into the solid-phase purification process to support its intended purpose. The use of the riboside phosphoramidite 6 led to the incorporation of an ethyl phosphate triester into the DNA sequence intended to be released from the capture solid support 12.…”

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards

“…High-throughput approaches using an aminopropylated silica gel and chemoselective ligation are also being formulated for purifying synthetic DNA from solid-state supports. 83,84 Most DNA synthesizers allow for switching between a range of 0.05, 0.1, 0.2, 1, and 10 μmole synthetic scales with available commercial reagents including the CPGs matched to these. 85 Phosphoramidite chemistry also comes with the inherent benefit that trityl removal during deprotection step can be monitored either by absorbance or electrochemistry to give a real-time readout of the efficiency of each step and an estimate of the final yield.…”

Purification of Self‐Assembled DNA Tetrahedra Using Gel Electrophoresis

“…These methods include the (i) cooperative assembly of individual DNA strands (Wang & Seeman, 2007), (ii) hierarchical assembly of DNA motifs into larger structures (He et al., 2008), (iii) DNA origami method to fold a long piece of DNA into different shapes (Rothemund, 2006), and (iv) DNA brick strategy using single‐stranded DNA blocks (Ke, Ong, Shih, & Yin, 2012). With developments in high‐throughput chemical synthesis of DNA (Grajkowski, Cieślak, & Beaucage, 2017; Tian, Ma, & Saaem, 2009) and different chemical strategies for functionalization (Madsen & Gothelf, 2019), DNA nanostructures have found applications in different fields such as biosensing, drug delivery, bioimaging, nanoparticle assembly, molecular computation, and synthetic gene circuits (Hu, Li, Wang, Gu, & Fan, 2019; Scalise & Schulman, 2019; Xiao et al., 2019). For some applications, the DNA nanostructures need to be purified from intermediate byproducts (improperly assembled structures) and excess single strands.…”