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Methods of error filtration and correction post-gene assembly are a major bottleneck in the synthetic biology pipeline. Current oligonucleotide purification strategies, including polyacrylamide gel electrophoresis and high-performance liquid chromatography, are often expensive and labor-intensive, give low mass recovery, and contain hazardous chemicals. To circumvent these limitations, we explored an enzymatic means of oligonucleotide purification using RecJ, which is the only known exonuclease to digest single-stranded DNA (ssDNA) in the 5' to 3' direction. As a potential application to remove failure strands generated in oligonucleotide synthesis, we found RecJ does not recognize the 5' dimethoxytrityl blocking group and could therefore be used to specifically target and digest unblocked failure strands. In combination with ssDNA binding protein (SSBP), which acts to recruit RecJ via C-terminal recognition, secondary structure formation is precluded, allowing for enhanced RecJ processivity. Using this method to purify crude trityl-on oligonucleotides, we also found on average 30 units of RecJ with 0.5 μg of SSBP digests 53 pmol of 5' hydroxylated ssDNA (60 min at 37 °C). With these parameters, the average purity is increased by 8%. As such, this novel method can be adapted to most laboratory practices, particularly those with DNA synthesis automation as a simple, inexpensive (<$4), and eco-friendly means of oligonucleotide trityl-on purification.

Solid phase spermine oligomerization via guanidine linkers was achieved using activated thiourea coupling reaction with primary amino group. Disymmetric spermine synthon was efficiently synthesised in eight steps from spermine. MMT group was used as coupling monitor and resulting oligomeric spermines were conjugated to oligonucleotides., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We previously reported a series of new amino-linkers, consisting of an aminoethyl carbamate structure (Komatsu, 2008). We have now examined the chemical properties of oligonucleotides modified with an ssH-linker, which is the simplest and most cost-effective derivative of the series. Although it was previously shown that monomethoxytrityl protection on a primary amine of the ssH-linker was cleaved under weakly acidic conditions (1% acetic acid), we found that the deprotection also proceeded in aqueous buffer solutions (pH 6.0, 7.0). The MMT group was removed much faster than other commercially available amino-linkers, and this property enabled the ssH-modified oligonucleotides to be conveniently purified with a cartridge column. Furthermore, the ssH-modified oligonucleotides were utilized in on-support labeling reactions. As compared with other amino-linkers, the ssH-linker was superior in terms of its purification and reaction efficiencies.

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.

We report herein the design, preparation and first applications of novel trityl tags with adjustable stability, efficient as protecting groups or MS analytes.

Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr-C-phosphonate (DMTr=4,4'-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr-C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr-C-phosphonate moiety located internally or at the 5'terminal hydroxy group. DMTr-C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.

This unit provides strategies on the maximization of synthetic yield, the generation of sequences containing site-specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection of DNA and RNA oligonucleotides.

Protocols are given for purification of oligonucleotides by dimethoxytrityl-sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean-up by selective adsorption, and elution on solid-phase media. Many of these products are prepackaged, single-use cartridges or columns filled with affinity or size-exclusion media.

The cyanoborane (-BH2CN) nucleosides and nucleotides are a new class of compounds that mimic natural and synthetic congeners in many ways and exhibit interesting biochemical and biophysical properties. The B-N bond is isoelectronic with the C-N+ bond of N7-alkylated 2'-nucleosides, as well as the C-C bond of naturally occurring 7-alkyl-7-deazanucleosides. These compounds differ from normal guanosine in that they are incapable of hydrogen bonding at the 7-position. The syntheses of N7-cyanoborane 2'-deoxyguanosine, N2-(dimethylaminomethylene)-N7-cyanoborane 5'-(dimethoxytrityl)-2'-deoxyguanosine (3), and N2-isobutyryl-N7-cyanoborane 5'-(dimethoxytrityl)-2'-deoxyguanosine (9) are described. Removal of the dimethoxytrityl (DMT) group from 3 or 9 is accompanied by significant loss of the cyanoborane moiety. Additionally, dimethoxytritylation of a cyanoboronated nucleoside leads to partial deboronation, thus limiting use of the commercially available 5'-DMT nucleosides as viable precursors in base-boronated oligonucleotide synthesis. The incompatibility of the cyanoborane moiety under DMT removal/addition conditions necessitated the search for an alternative method of protecting the 5'-hydroxyl of the nucleoside. This paper addresses the possible cause of deboronation and describes the synthesis of N7-cyanoboronated nucleosides by a method that avoids transient protection of the sugar hydroxyls.