Cyanine functionalised solid supports for oligonucleotide synthesis

Sheena Aitken, Jennifer Mathieson, Grant McGeoch, Catherine McKeen and Joanna Wrzesien; Link Technologies Ltd, Bellshill, UK. jennifer@linktech.co.uk

Introduction
Cyanine dyes are used as fluorescent markers in oligonucleotide synthesis,1 primarily for molecular diagnostics such as the preparation of probes used in monitoring real-time PCR, fluorescence in situ hybridisation (FISH) and in SERRS based DNA detection assays. Their emission spectra can be tuned by altering the length of the polymethine chain and solubility in organic or aqueous solvents can be altered via the substituents on the aromatic ring. Cyanine dyes absorbing over 540nm and 650nm have been studied. These dyes are commercially available as phosphoramidites under the names Cy3 (1) and Cy5 (2) respectively, usually for incorporation onto the 5 end of an oligonucleotide during solid phase synthesis, although there is the possibility of including it within the sequence of nucleotides as it has a MMTr-protected hydroxyl group in addition to the phosphoramidite. This, however, is not common 3 4 due to the lack of heterocyclic base in their structure and as such they do not have the ability to participate in base pairing. Internal addition can therefore destabilise any duplexes formed. We have attached these dyes onto CPG supports (3, 4) in order to allow direct incorporation onto the 3 end of an oligonucleotide. Previously this was done by adding the dye post-synthetically onto an amino-modified oligonucleotide or by adding the amidite to a support functionalised with a modification that will not interfere with the use of the oligonucleotide (e.g. phosphate, spacer). This will be especially useful for functions such as detecting DNA-protein interactions2 i.e. for the production of double-labelled probes for use in fluorescence resonance energy transfer (FRET) studies. 1 2

Experimental
A series of short (20-21mer) oligonucleotides were synthesised in order to compare the use of Cyanine 540-functionalised support 3 with the phosphoramidite 1. Modifiers in phosphoramidite form were used in the following sequence, where X is the phosphoramidite modifier. TTT TTT TTT TTT TTT TTT TXT CPG functionalised with the modifier were used in the following sequence, where X is the modifier attached to the CPG. TTT TTT TTT TTT TTT TTT TX Oligonucleotides were synthesised on an ABI394 DNA/RNA synthesiser using 1000 CPG solid support and amidites at 0.1M concentrations in acetonitrile.

Synthesis conditions
The following reagent specifications were used: activator - 0.25M ETT; cap A - THF/pyridine/acetic anhydride (8:1:1); cap B - 10% methylimidazole in THF; oxidiser - 0.02M iodine in THF/pyridine/ water (7:2:1); deblock - 3% TCA/DCM. 30s coupling times were used for DNA amidites and 6min coupling times for cyanine dye modifications.

Analysis Conditions
LCMS was carried out on an Agilent 6220 Accurate Mass TOF LCMS with 1260 pumps and DAD, using a Zorbax C18, 3.5m, 2.1x30mm column, Buffer A: 190mM HFIP, 7mM TEAA, 5% MeOH in water, Buffer B: MeOH, with a gradient of 0-100% B over 12min. All samples were at a concentration of 2OD/mL. RP-HPLC on all oligonucleotides was carried out using a Waters X-Bridge OST C18, 2.5m, 4.6x50mm column, Buffer A: 0.1M TEAA, Buffer B: MeCN over a gradient of 0-50% B over 15min at a flow rate of 1mL/min.

Deprotection Conditions
All oligonucleotides were cleaved and deprotected using AMA at room temperature for 2h then immediately passed through a G25 column.

Results
HPLC data for the oligonucleotides synthesised from Cyanine 540-functionalised support are comparable in purity by crude HPLC to the oligonucleotides synthesised from Cyanine 540 phosphoramidite (Cy3) (see Figures 1 and 2). A similar purity is also observed in LCMS analysis of the crude oligonucleotide with the correct mass being identified (see Figures 3 and 4).
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Conclusions
Initial results show that incorporating the cyanine dyes at the 3 end of an oligonucleotide gives comparable results to them being incorporated in phosphoramidite form. The greatest advantage of the cyanine dye functionalised support is that it eliminates the requirement of the use of an amino linker followed by addition of the dye by post-synthetic labelling. Similar results have been observed for comparison of Cyanine 650 phosphoramidite (Cy5) (2) and Cyanine 650-functionalised CPG (4).

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Further Information
For further information please contact Dr Catherine McKeen,
Figure 3. LCMS data of Cyanine 540 CEPA (Cy3) oligo.

1000

Technical Manager, Link Technologies (catherine@linktech.co.uk) or Jennifer Mathieson, Senior Chemist, Link Technologies Ltd (jennifer@linktech.co.uk).

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Calc. 6862.33, Obs. 6833.56

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1.

Fluorescence based strategies for genetic analysis, R.T.

Ranasinghe and T. Brown, Chem. Commun., 5487-5502, 2005.

Figure 1. RP-HPLC data of Cyanine 540 CEPA (Cy3) oligo.

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1000 900 800 700 600

Fluorescence resonance energy transfer in near-infrared

fluorescent oligonucleotide probes for detecting protein-DNA interactions, S. Zhang, V. Metelev, D. Tabatadze, P.C. Zamecnik, A. Bogdanov Jr, Proc. Natl. Acad. Sci. USA, 105, 4156-4161, 2008. Cy3 and Cy5 are trademarks of GE Healthcare. www.linktech.co.uk

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Figure 4. LCMS data of Cyanine 540 CPG oligo. Calc. 6558.29, Obs. 6529.55.

Figure 2. RP-HPLC data of Cyanine 540 CPG oligo.