Oligonucleotide, synthesis

Two opposing trends have become apparent in the field of oligonudeotide synthesis on the one hand, the development of methods and equipment has been such that operators do not now require so much chemical expertise, and on the other, that the enormous growth in the need for oligonucleotides has made it more usual, and economical, to obtain these materials as custom synthesised products commercially. 

The most important nudeic add modification is undoubtedly radioactive labelling. 

Some of these reactions can also be used for non-radioactive labelling (Isaac 1994) since Tag-polymerase, DNA polymerase I and also terminal transferase can accept compounds such as digoxygenin-dUTP (DIG-dUTP) and the dideoxy derivative DIG-ddUTP as substrates. DIG-labelled DNA can be detected colorimetrically by antibodies coupled to peroxidase using chromogenic substrates. Similar procedures can also be used to label RNA using DIG-UTP with T7- or SP6-RNA polymerases. Digoxygenin labelling kits are commercially available. 

Reagent Controller Herringbone Binary Tree Column Array 

Each column has a flush valve that opens and closes in unison to allow residual reagent in the binary tree to be washed away between steps to avoid contamination. The addressable inlet valve controls which columns the reagents enter. The compression valves prevent rearrangement of closely packed spherical beads when a pressure drop occurs between steps [36]. Source Copyright 2010 Oxford University Press. 

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Oligonucleotide Synthesis (phosphoramidite method - Lessinger) Review Tetrahedron 1992, 48,2223... 

In step 1 of each oligonucleotide-synthesis cycle the 5 -terminal 4,4 -dimethoxytrityl protecting group is removed with trichloroacetic acid, and the support is washed with acetonitrile to prevent dctritylation of the next incoming phosphoramidite. The 4,4 -dimethoxy-... 

The protected nucleoside-3-phosphoramidite monomer units such as 671 are used in the solid-phase oligonucleotide synthesis. In the 60mer synthesis, 104 allylic protective groups are removed in almost 100% overall yield by the single Pd-catalyze reaction with formic acid and BuNH2[432], N,(9-protection of uridine derivatives was carried out under pha.se-transfer conditions[433]. 

Oligonucleotide synthesis involves specialized blocking and coupling reactions the chemistry of which is beyond the scope of a typical introductory course The in terested reader is referred to http //WWW bi umist ac uk/ users/dberrisford/1 MBL/ nucleicacidB html... 

This substantial group was developed as a fluorescent, acid-labile protective group for oligonucleotide synthesis. It has properties very similar to those of the DMTr group except that it can be detected down to 10 M on TLC plates with 360-nm ultraviolet light. 

Synthetic organic polymers, which are used as polymeric supports for chromatography, as catalysts, as solid-phase supports for peptide and oligonucleotide synthesis, and for diagnosis, are based mainly on polystyrene, polystyrene-divinylbenzene, polyacrylamide, polymethacrylates, and polyvinyl alcohols. A conventional suspension of polymerization is usually used to produce these organic polymeric supports, especially in large-scale industrial production. 

The Dnseoc group was developed as a base-labile protective group for the 5 -hydroxyl in oligonucleotide synthesis. It is cleaved with DBU in aprotic solvents. The condensation of oligonucleotide synthesis can be monitored by UV detection at 350 nm or by fluorescence at 530 nm of the liberated vinylsulfone. ... 

The steps involved in automated oligonucleotide synthesis illustrate the current use of protective groups in phosphate chemistry (Scheme 1). Oligonucleotide synthesis involves the protection and deprotection of the 5 -OH, the amino groups on adenine, guanine, and cytosine, and -OH groups on phosphorus. 

The disadvantage of this method is that the dichloridites and monochloridites are sensitive to water and thus could not be used readily in automated oligonucleotide synthesis. This problem was overcome by Beaucage and Caruthers, who developed the phosphoramidite approach. In this method, derivatives of the form R 0P(NR2)2 react with one equivalent of an alcohol (catalyzed by species such as l//-tetrazole) to form diesters, R OP(OR")NR2, which usually are stable, easily handled solids. These phosphoroamidites are easily converted to phosphite triesters by reaction with a second alcohol (catalyzed by l//-tetrazole). Here, again, oxidation of the phosphite triester with aqueous iodine affords the phosphate triester. Over the years, numerous protective groups and amines have been examined for use in this approach. Much of the work has been reviewed. ... 

This group, used for 5 -phosphate protection, has hydrophobicity similar to that of the dimethoxytrityl group and thus was expected to assist in reverse-phase HPLC purification of product from failure sequences in oligonucleotide synthesis. The group is cleaved with Bu4N F in DMSO at 70°. ... 

The trifluoroethyl group was used as an activating group in the phosphotriester approach to oligonucleotide synthesis, as well as a protective group that could be removed with 4-nitrobenzaldoxime (tetramethylguanidine, dioxane, H20). ... 

The rate of oligonucleotide synthesis by the triester method using mesitylenesul-fonyl chloride was increased five- to tenfold when this group was used as a protective group during intemucleotide bond formation. It was removed with coned. NH4OH at 60° for 12 h or by the oximate method. ... 

This lipophilic group, developed for 5 -phosphate protection in oligonucleotide synthesis, is removed with 80% AcOH in 1 h. The related trityloxyethylamino group has been used in a similar capacity for phosphate protection and is also cleaved with 80% AcOH. ... 

Morpholine has been used for 5 -phosphate protection in oligonucleotide synthesis and can be cleaved with 0.01 N HCl without significant depurination of bases having free exocyclic amino functions. 

Silica or porous glass is usually used as the solid phase in oligonucleotide synthesis. The support is functionalized through an amino group attached to the silica surface. There is a secondary linkage through a succinate ester to the terminal 3 -OH group. 

Although use of automated oligonucleotide synthesis is widespread, work continues on the optimization of protecting groups, coupling conditions, and deprotection methods, as well as on the automated devices.56... 

Warren, W. and Vella, G., Analysis and purification of synthetic oligonucleotides by high-performance liquid chromatography, in Oligonucleotide Synthesis Protocols, Agrawal, S., Ed., Humana Press, Totowa, NJ, 1993, 235. 

Nucleotides and nucleic acids are critical tools in the areas of gene expression, therapeutics, and diagnostics. However, there are certain challenges associated with their large-scale purification and subsequent characterization. While solid-state oligonucleotide synthesis is relatively simple and can be totally automated, intra- and intermolecular associations may occur involving shorter sequences that may hybridize with the desired full length... 

On use of N,N -dicyclohexylcarbodiimide instead of sulfonyl chlorides as condensation reagent in oligonucleotide synthesis, then the pyro-, tri- and tetraphosphate stages are again involved 124). The metaphosphate 183 a is found in small amounts by 3iP-NMR spectroscopy, but again no cyclic trimetaphosphate 184 can be detected, which would also be a possible phosphorylation reagent. 

It is still unclear how much general validity attaches to the metaphosphate mechanism as formulated for the present variant for the oligonucleotide synthesis. Depending upon the reaction conditions, preparative method, activating reagent, and length of the nucleotide block, other mechanisms may dominate. 

The mechanism of the Li-Nicolaou experiment shows the manifold possibilities for oligonucleotide synthesis. However, this replication system is limited to special... 

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