It is widely known that branching monomers are used in the preparation of oligonucleotide dendrimers.1,2,3
Link Technologies offers one such monomer; Branching Me-dC-CE Phosphoramidite (2150). This has been used to generate comb and fork like oligonucleotide structures for use in nucleic acid hybridisation assay as a means of signal amplification.4
However, it is also possible to incorporate reporter groups into specific sites within an oligonucleotide sequence using this monomer in an analogous manner to the method reported by Brown et. al.5 Here they used a branching dT phosphoramidite (1) to incorporate dyes such as Cyanine 5 (2521) within the sequence for use in real-time probes such as HyBeacon or Angler probes.
In this case 1 was incorporated within an oligonucleotide where the 5’-end is blocked either by retaining the DMT group, capping with acetyl protection or by the incorporation of a terminal modifier e.g. 6-FAM CE Phosphoramidite (2134). While keeping the oligonucleotide on the column, the Fmoc group is removed with 20% piperidine in MeCN or DMF and the cyanine dye phosphoramidite is added to the branching point under the same conditions as incorporation at the 5’-end. This is outlined in Figure 1.
Figure 1. Use of Fmoc Protected Branching dT amidite for incorporation of cyanine 5.
Although it is possible to incorporate 2521 within an oligonucleotide sequence, this results in a destabilised duplex whereas the use of the modified dT has no adverse effect.
This is also true when 2150 is used in the same way. Although a Me-dC anaolgue, the presence of the branching chain on the N4 position of the pyrimidine results in this modifier having hybridisation properties akin to dT rather than dC hence is incorporated as a ‘dT’ position of the oligonucleotide sequence. In this case (see Figure 2), the levulynyl protection is removed using 0.5M hydrazine hydrate in pyridine/acetic acid 1:1.
Figure 2. Use of Levulinyl Protected Branching MedC amidite for incorporation of cyanine 5.
The use of such branching monomers opens up the possibility of incorporating modifiers only available as 5’-addition amidites internally within the sequence. For instance this gives a means of generating HEX-dT (2) using 2150 and 2136 or cholesteryl dT (3) using 2150 and 2170 as shown in Figure 3. Neither of these dT modifiers are commercally available as amidites.
Figure 3. Generation of HEX dT (2) and cholesteryl dT (3) within an oligonucleotide sequence using 2150.
This can be particularly useful in evaluating which marker works best in a given application without the expense of synthesising a range of modified dT amidites to get the same result. Using this information, the prefered modified dT amidite can be synthesised with the peace of mind of knowing that the resulting oligonucleotide will give the desired result when used in an assay.
Although originally 2150 was designed for the preparation of highly branched oligonucleotides at Link we see the potential of this product to allow the incorporation of modified dT bases not commercially available within the sequence.
If you require further information on this article please do not hesiate to contact us.
- Oligonucleotide dendrimers: Synthesis and use as polylabelled DNA probes, M. S. Shchepinov, I. A. Udalova, A. J. Bridgman and E. M. Southern, Nucleic Acid Research, 1997, 25(22), 4447-4454. Full Text PDF
- An improved divergent synthesis of comb-type branched oligodeoxyribonucleotides (bDNA) containing multiple secondary sequences, T. Horn, C.-A. Chang and M. S. Urdea, Nucleic Acid Research, 1997, 25(22), 4835-4841. Full Text PDF
- Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter, P. A. Olsen, C. McKeen and S. Krauss, Gene Therapy, 2003, 10, 1830-1840. Abstract
- Forks and combs and DNA: the synthesis of branched oligodeoxyribonucleotides, T. Horn and M. S. Urdea, Nucleic Acid Research, 1989, 17(17), 6959-6967. Full Text PDF
- Synthesis of a modified thymidine monomer for site-specific incorporation of reporter groups into oligonucleotides, L. J. Brown, J. P. May, T. Brown, Tetrahedron Letters, 2001, 42(13), 2587-2591. Abstract