Introduction The advantage of chemically synthesized oligonucleotides is the possibility of creating the artificial molecules that can be used in specific application. The increasing utilization of synthetic oligonucleotides in genetic analysis, diagnostic, and therapeutic applications in post genomics area has stimulated the development of a variety of reagents for the functionalization of oligonucleotides. Retrogen offers most of these modifications.
Phosphorylation
Oligonucleotides can be phosphorylated enzymatically at the 5'-terminus by polynucleotide kinase. This reaction is reversible, and is difficult to control. For applications, like in gene assemblage, where complete phosphorylation is important, it is advisable to introduce the phosphate chemically. This can be achieved easily by using a chemical phosphorylating amidite at the last condensation cycle. The following phosphorylation is available at Retrogen:
Modifications
Available Positions
5'-Phosphorylation
5'
3'-Phosphorylation
3'
Amination The purpose of terminal amination is to introduce a non-hindered nucleophilic centre for the specific introduction of further functionalization with any desired group (e.g. biotin, fluorescein, rhodamine, Texas Red, digoxigenin, alkaline phosphatase, horseradish peroxidase). This primary amine reacts with N-hydroxy-succinimide esters (NHS esters) to form amide bonds. The terminal amino groups will react with mild nucleophilic reagents, approx. 100 times faster than the heterocyclic aminogroups at the bases. The reaction is favored at alkaline pH , because the primary amine is kept in an unprotonated state. The following are 5'-Amino-Modifers available at Retrogen:
Modifications
Available Positions
5'-Amino-Modifier C6
5'
5'-Amino-Modifier C12
5'
5'-Amino-Modifier 5
5'
5'-Amino-Modifier C3-TFA
5'
5'-Amino-Modifier C6-TFA
5'
Amino Modifier C2 dT
5', Internal
Amino Modifier C6 dT
5', Internal
Amino Modifier C3
3'
Amino Modifier C7
3'
Spacer Arm The spacer phosphoramidite C16 attaches a lipophilic group to the 5'-terminus improving cellular uptake of oligonucleotides and therefore biological activity of antisense oligonucleotides. The following are spacers available at Retrogen:
Modifications
Available Positions
dSpacer
5', Internal
Spacer 18
5', Internal
Spacer 9
5', Internal
Spacer C12
5', Internal
Spacer C3
5', Internal, 3'
Thiolation Thiolation can be used as an alternative to amination. Furthermore, it opens the possibility of specific coupling to a sulphydryl containing ligand through disulphide bridge. The thiol group is protected by a trityl group and separated by a six-carbon linker from the amidite group. The protecting trityl can be removed by treatment with silver nitrate and dithiothreitol. The following are 5'-Thiol-Modifiers available at Retrogen:
Modifications
Available Positions
Thiol Mod C3 S-S
3'
Thiol Modifier C6 S-S
5'
Biotin Biotinylated oligonucleotides have been used in a large number of molecular biology applications including quantification of PCR-amplified sequences, chemiluminescent sequencing, in situ hybridization, solid phase restriction site mapping, single base mutational analysis, genomic walking, and cloning of unknown DNA sequences. Once incorporated, the biotin label can be detected by standard streptavidin-based detection methods.
Modifications
Available Positions
Biotin-TEG
3', 5', internal
Biotin-dT
Internal
Fluorescein
Fluorescein is introduced for direct fluorescence detection. Fluorescein-labelled oligonucleotides have been used for non-isotopic fluorescent sequencing, PCR quantitation, colour PCR, and studies of cellular uptake and distribution of antisense olgonucleotides. The fluorescein label can be used either as a hapten for antibody binding or as a fluorophore for fluorescent signal generation.
Modifications
Available Positions
TAMRA
3', 5'
6-FAM
3', 5'
Cy3
5'
Cy5
5'
Fluorescein
3', 5'
Fluorescein-dT
3', 5', internal
HEX
5'
TAMRA-dT
5', internal
TET
5'
Phosphorothioate (S-oligo) Phosphorothioate analogues of DNA and RNA have sulphur in place of oxygen as one of the non-bridging ligands bound to the phosphorus. Phosphorothioates have been shown to be more resistant to nuclease degradation than the natural DNA and RNA and still to bind to complementary nucleic acid sequences. Phosphorothioate oligodeoxy-nucleotides have demonstrated their usefulness as antisense molecules inhibiting gene expression and as potential chemotherapeutic agents. Phosphorothioate is available at any position in an oligonucleotide, and can be used multiple times within a sequence.
DeoxyInosine
This is a deoxynucleoside with the base hypoxanthine (6-hydroxypurine). When used within a hybridization probe, deoxyinosine residues can form base-pairs with dA, dC, dG, or T residues on the target strand. DeoxyInosine can be used instead of wobbles and has the advantage that the hybridization probe is not "diluted" by the non-pairing components of the wobbles. DeoxyInosine is available at any position in an oligonucleotide, and can be used multiple times within a sequence.D
Degenerate Bases (Wobbles)
For some experiments the synthesis of sequences which have one or more degenerate positions may be required. This can be achieved using a mixture of the required phosphoramidites (2, 3 or 4 mixed together) in the addition step at the appropriate position. The resulting oligonucloetide will have a mixture of bases at the specified position in a ratio similar (but not equal) to the proportions in which the bases were mixed. When the desired redundancy is located at the 3'-end, a mixture of supports is used.
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