*****Glen Research Glen Report*****
5' LABELLING - FLUORESCEIN AND CYANINE DYES, BIOTIN
Traditional radioactive labelling of oligo-nucleotides is being
rapidly supplanted by non-isotopic procedures. Increased awareness of
potential operator exposure to radiation, along with the increasing
cost of proper disposal of radioactive waste, has clearly mandated
some change. At the same time, diagnostic procedures using
non-isotopically labelled oligonucleotides can be carried out in a
much wider variety of testing laboratories. Currently,
non-radioactive labelling is broken into two main segments: labelling
using fluorescent tags which are detected directly at their emission
wavelength; and labelling using tags like biotin or digoxigenin which
are detected indirectly after capture or association with a suitably
substituted enzyme or antibody.
Since the majority of oligonucleotides are destined for sequencing
and amplification experiments, which require the 3'-terminus to be
available for extension, labelling of the 5'-terminus is the most
popular technique. While our future development of new labels will
concentrate on 5' labelling, it would be imprudent of us to neglect
labelling of the 3' terminus which can be especially useful in probe
applications. However, the primary focus of this article is on a
series of new 5' labels, including fluorescent and non-fluorescent
tags shown in Figure 1.
Fluorescein labelled oligonucleotides have found applications in
DNA sequencing and amplification, as well as techniques for genetic
analysis. In the forefront of this development has been our current
fluorescein phosphoramidite, Figure 1 (1), which has proved to be a
popular, versatile and effective reagent for the preparation of
labelled oligonucleotides. Using this product or the related support
(2), fluorescein molecules may be placed anywhere within the
sequence. Although the branched structure of the spacer allows a
degree of versatility, it can cause some minor complications in use.
Due to steric hindrance of the phosphoramidite, an increased coupling
time of 15 minutes is optimal. Also, purified oligonucleotides
labelled with this product exhibit multiple peaks, especially in
reverse phase (RP) HPLC, even though the product is prepared from the
5-carboxyfluorescein isomer alone. (Fluorescein's interesting
numbering system is detailed in Figure 2.) This multiplicity is the
result of diastereomer formation due to the chiral center in the
spacer structure, along with the standard pH dependent structural
variation of fluorescein as shown in Figure 2. A typical RP HPLC
profile of an oligonucleotide labelled with this product is shown in
Figure 3. Although not problematical in the usage of the labelled
oligonucleotide, a product containing multiple peaks is always
worrisome to customers. We are happy to maintain the supply of this
product but clearly a fluorescein phosphoramidite with a simpler
structure designed only for 5' labelling would be desirable.
And so we introduce the 5'-fluorescein phosphor-amidite (3) whose
sole role is to label the 5'-terminus during oligonucleotide
synthesis. The product contains no 4,4'-dimethoxytrityl (DMT) group
and can be added only once at the 5'-terminus, thereby terminating
synthesis. Although this product is prepared using the
6-carboxyfluorescein derivative, its spectral characteristics are
identical to those derived from product (1) in Figure 1.
5'-Fluorescein phosphoramidite can be used with the standard cycle of
all DNA synthesizers. However, as with all minor bases and most
labelling reagents, it will benefit from an extended coupling time of
To take advantage of the multicolor detection capability of modern
DNA sequencers and genetic analyzers, further derivatives of
5'-fluorescein phosphor-amidite with shifted absorbance and emission
maxima would obviously be of interest. We are therefore happy to
introduce the tetrachloro (4) and hexachloro (5) phosphoramidites as
the first two in a series of fluorescein analogues. The use of these
products is identical to their fluorescein parent, as described
above. The spectral characteristics of these dyes are detailed in
Table 1 and typical RP HPLC traces are shown in Figure 4.
The fluorescein structure is very compatible with oligonucleotide
synthesis because it is resistant to hydrolysis with ammonium
hydroxide, even when elevated temperatures and extended deprotection
times are used.
Unfortunately, the same does not apply to rhodamine derivatives which
are not sufficiently stable to survive conventional deprotection.
These must be attached to amino-modified oligonucleotides using
post-synthesis labelling techniques. Typically, an activated
carboxylate, usually N hydroxysuccinimide (NHS) ester, of the dye in
solution in DMF or DMSO is conjugated with the amino-modified
oligonucleotide in sodium carbonate/bicarbonate buffer at pH 9.
Although this technique is time consuming and places demands on the
final purification to remove unconjugated dye, it is nevertheless
routine and successful.
We feel that our role at Glen Research is to offer interesting
products as phosphoramidites but will offer the most popular
rhodamine derivative (tetramethylrhodamine, TAMRA) as an NHS ester
(6) as an interim step.
Some of our customers have expressed an interest in labelling with
cyanine dyes, as well as admiration for their performance in labelled
oligonucleotides. The commercial ownership of these dyes is a story
in itself but we are happy to introduce them here (see SMALL PRINT
page 4) and hope to maintain supply while stimulating development of
new uses for them. The two cyanine derivatives we are introducing are
Cy3tm (7) and Cy5tm (8) which
differ in structure simply by the number of carbons in the conjugated
Although these products have a 4-monomethoxytrityl (MMT) group and,
in principal, could be added in multiple additions, we expect them to
be added only once to the 5'-terminus. The MMT group should therefore
be removed on the synthesizer. The absorbance of the MMT cation
(yellow) is noticeably different from the DMT cation (orange), and
so, absorbance based trityl monitors will detect it incorrectly as a
low coupling. On the other hand, conductivity detectors will
interpret the release more correctly.
Deprotection of oligos containing Cy3 and Cy5 may be carried out with
ammonium hydroxide at room temperature, regardless of the base
protecting groups on the monomers used. If there is a need to use
ammonium hydroxide at elevated temperature, Cy3 is more stable than
Cy5, but it is always prudent to use monomers with base labile
protecting groups to limit the exposure time to 2 hours or less at
55°C. Typical RP HPLC traces of Cy3- and Cy5-labelled
oligonucleotides are shown in Figure 5.
Another biotin phosphoramidite, when Glen already has three such
monomers available! Why?
It is fair to say that we have had considerable interest in our new
5' biotin phosphoramidite (9) for a long time. However, it is covered
by patent and, although we could have purchased the product and
offered it under license, we were totally dissatisfied with the
purity of the available product.
We have now obtained manufacturing rights to this product and we can
at last offer this 5'-biotin phosphoramidite under license (see SMALL
PRINT). While the product which was available previously clearly
performed well, the chromatographic purity was substantially less
than 90%. We feel that 90% should be the absolute minimum purity for
a quality product and the 5'-biotin phosphoramidite from Glen
Research will always meet this specification. Higher purity should
translate into constant performance with better lifetime on an
instrument once the product is dissolved.
Why has this particular biotin phosphoramidite interested us so much?
Its performance features are listed below:
- It is, of course, freely soluble in acetonitrile.
- It can be used with the standard cycle of all instruments
(although it will benefit from up to a 3 minute coupling
- Because of the short coupling time, it is more robust than our
other biotin products with better performance in higher moisture
situations and, therefore, has a better lifetime on the
- Because of the DMT group on the biotin, the coupling yield can
still be determined on the synthesizer.
- CAUTION! 5'-Biotin phosphoramidite can be added only once to
the 5'-terminus of an oligonucleotide. However, the DMT group on
the biotin can be used in RP cartridge and HPLC purification
For reference, in Table 3 on the previous page, we compare,
contrast and suggest usage for our team of biotin labels.
All of the products described above are amenable to purification
using RP cartridge and HPLC techniques. Poly-Pak procedures are
provided with each product on delivery. They are also available for
review or download on the worldwide web:
Cy3 and Cy5 are trademarks of Amersham PLC.
5'-Biotin Phosphoramidite is sold under license from Zeneca PLC.
Poly-Pak is a trademark of Glen Research Corporation.
NHS Ester (Solution in anhydrous DMSO)