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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.
| Modifications |
Available
Positions |
| A
= Adenosine |
3',
5', internal |
| C
= Cytidine |
3',
5', internal |
| G
= Guanosine |
3',
5', internal |
| T
= Thymidine |
3',
5', internal |
| U
= Uracil |
3',
5', internal |
| K
= G or T |
3',
5', internal |
| M
= A or C |
3',
5', internal |
| R
= A or G |
3',
5', internal |
| S
= G or C |
3',
5', internal |
| W
= A or T |
3',
5', internal |
| Y
= C or T |
3',
5', internal |
| B
= C, G or T |
3',
5', internal |
| D
= A, G or T |
3',
5', internal |
| H
= A, C or T |
3',
5', internal |
| V
= A, C or G |
3',
5', internal |
| N
= any base |
3',
5', internal |
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