Purification Of Oligodeoxynucleotides Research Articles

Synthesis and Functionality Study of Photoswitchable Hydrazone Oligodeoxynucleotides.

This article provides a detailed procedure for the chemical synthesis and characterization of photoswitchable hydrazone phosphoramidite and its incorporation into oligodeoxynucleotides. The synthesis starts with commercially available deoxyuridine, followed by conversion of the 4-oxo into a 4-chloro moiety via Appel reaction to install the key hydrazone group in the absence of base. The hydrazone phosphoramidite building block is compatible with the conventional amidite chemistry protocols for solid-phase synthesis of oligodeoxynucleotides. Our method expands the current nucleotide pool by adding a novel, functional DNA building block that is suitable for a broad spectrum of applications, including the regulation of DNA-enzyme interactions and DNA synthesis by irradiation with cell-friendly blue light. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of photoswitchable hydrazone phosphoramidite Basic Protocol 2: Synthesis and purification of oligodeoxynucleotides containing the hydrazone photoswitch Basic Protocol 3: Primer extension assay for functionality studies of hydrazone cytidine.

Parallel, Large-Scale, and Long Synthetic Oligodeoxynucleotide Purification Using the Catching Full-Length Sequence by Polymerization Technique.

The catching by polymerization synthetic oligodeoxynucleotide (ODN) purification technique was shown to be potentially suitable for high throughput purification by purifying 12 ODNs simultaneously, to be convenient for large-scale purification by purifying at 60 μmol synthesis scale, and to be highly powerful for long ODN purification by purifying ODNs as long as 303-mer. LC-MS analysis indicated that the ODNs purified with the technique have excellent purity.

Polymerizable phosphoramidites with an acid-cleavable linker for eco-friendly synthetic oligodeoxynucleotide purification

Purification at the hundred milligram scale is as convenient as at the sub-milligram scale. Long sequences are easily isolated from complex mixtures. The waste to product ratio is reduced by hundreds of times over known methods.

A Highly Convenient Procedure for Oligodeoxynucleotide Purification

Purification of synthetic oligodeoxynucleotides (ODNs) is simply achieved by capping failure sequences with a polymerizable phosphoramidite followed by polymerization. In this article, the reduction of the amount of polymerization monomer to drastically increase ODN extraction efficiency, the use of a centrifugal filterunit to ease the extraction process and the notification of using fresh phosphoramidite solutions are described. In addition, further evidence to support the purity of ODN and discussions of ODN stability under radical polymerization conditions are provided.

Synthetic oligodeoxynucleotide purification by capping failure sequences with a methacrylamide phosphoramidite followed by polymerization

Synthetic oligodeoxynucleotides are simply purified by capping failure sequences with a methacrylamide phosphoramidite, co-polymerization with N,N-dimethylacrylamide and extraction with water.

Synthetic 5′-phosphorylated oligodeoxynucleotide purification through catching full-length sequences by polymerization

The readily scalable catching by polymerization purification technology has been further advanced to purify 5′-phosphorylated synthetic oligodeoxynucleotides (ODNs). The new technology utilizes a phosphoramidite that contains a fluoride-cleavable diisopropylsilyl acetal linker and a polymerizable methacrylamide group, and is capable of phosphorylation of ODN. For purification, the phosphoramidite was coupled to the 5′-end of full-length ODN on a synthesizer. Because failure sequences were capped in each synthetic cycle, only the full-length sequences were phosphinylated and acrylated. After cleavage and deprotection, the crude ODN was subjected to polymerization under typical acrylamide gel formation conditions. The full-length ODN was incorporated into polymer. The failure sequences and other impurities were simply removed by washing with water. Pure full-length ODN that contained a 5′-phosphate group was cleaved from the polymer with HF–pyridine. Reversed-phase (RP) HPLC showed that the ODN was pure, and the recovery yield was higher than that of typical preparative HPLC purification.

Synthetic oligodeoxynucleotide purification by polymerization of failure sequences

Synthetic oligodeoxynucleotide is purified by capping failure sequences with an acrylated phosphoramidite followed by polymerization and product extraction. The method is suitable for large scale oligonucleotide drug purification.

Scalable Synthetic Oligodeoxynucleotide Purification with Use of a Catching by Polymerization, Washing, and Releasing Approach

Synthetic oligodeoxynucleotides are purified with use of a catching by polymerization, washing, and releasing approach. The method does not require any chromatography, and purification is achieved by simple operations such as shaking, washing, and extraction. It is therefore useful for large-scale purification of synthetic oligonucleotide drugs. In addition to purification of oligonucleotides, this catching by polymerization concept is expected to be equally useful for purification of other synthetic oligomers such as peptides and oligosaccharides.

Chromatographic separation of oligodeoxynucleotides with identical length: Application to purification of oligomers containing a modified base

A general procedure is described for separation and purification of oligodeoxynucleotides of identical length but different base composition, in particular, of oligomers containing modified bases such as 4-substituted thymines and 6-substituted guanines, using an anion-exchange column (either Mono Q or NucleoPac). The modified oligomers can be well separated from the analogous oligomers containing unmodified thymine or guanine under the basic conditions of the chromatography. The effects of oligomer length, base composition, and lipophilicity on the separation are discussed. A general rule which can be used for prediction of the order of elution of different oligomers and for estimation of tautomeric form of a modified base in the oligomer is presented.

A New Combined Purification/Phosphorylation Procedure for Oligodeoxynucleotides

AbstractWe describe a new principle for the purification of oligodeoxynucleotides using a purification handle which leads to a covalent linkage of the desired DNA sequence to a solid matrix. Truncated sequences are removed by washing steps. The release of the pure target sequence is performed in such a way that it is obtained directly in its 5′‐phosphorylated form which represents a further advantage. In addition, the system can be used for 5′‐thiol modifications of synthetic DNA.