Branching Modification

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Applicable Product

2150 5-Me-dC-Brancher-CE Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

2150 C51H68N5O10P 942.10 403.37 2.65 5.31 10.61

General

Due to the possible complexity of these syntheses, we would advise customers to use these recommendations only as a guide and to optimise the conditions for their own use depending on the sequences and other modifiers employed.

Note that 3’-phosphate modification is not stable to the levulinyl deprotection step and the oligo is cleaved from the support.

Synthesis of simple branched structures

The product is simple to use in the synthesis of branched oligos with a small number of Branching Modifier (BM) inclusions (e.g. the “fork” structure created by using just one BM). The primary sequence is synthesised, incorporating the BM as follows:

Coupling

No changes are required from the standard method recommended by the synthesiser manufacturer. Coupling is as per standard nucleoside amidites.

Levulinyl Deprotection

If the secondary sequence is not required at the 5’ end, this must be capped prior to removing the levulinyl group. Prior to secondary sequence synthesis, the column is removed from the synthesiser and the levulinyl group is selectively removed without cleavage of the oligonucleotide by treatment with 0.5ml freshly-prepared 0.5M hydrazine hydrate in 1:1 pyridine/acetic acid (when using a 40nmol to 1μmol scale; use 10-15ml for 10μmol synthesis columns). To do this, fit the column with syringes and wash the solution back and forth. Allow to react for 15min (note sequences with many BM molecules require treatment for up to 90min to ensure complete levulinyl removal). Rinse the solid support with 10ml acetic acid/pyridine (1:1), followed by extensive rinsing with acetonitrile before drying under a stream of argon.

Secondary Sequence

At this point the column can be returned to the synthesiser to proceed with the secondary sequence synthesis. For primary sequences with only one or two BM molecules, the secondary sequence can be carried out using standard conditions although optimisation may be required.

It is important that no initial capping step is carried out in the synthesis cycle.

Note that for the secondary sequence, the equivalents of phosphoramidites must be increased to account for the number of growing chains. For example, if there are two branching points, double the molar equivalents of amidite will theoretically be required.

Cleavage & Nucleobase Deprotection

After secondary synthesis is complete, the oligonucleotide is cleaved from the support and base-deprotected using standard deprotection conditions (although this will be determined by other modifications within the oligo).

However, due to the complex secondary structures that can now form, ammonium hydroxide overnight at 55˚C gives the best results.

Synthesis of complex ‘comb’-like structures

Construction of more complex comb-like structures with many BMs in the primary sequence requires greater control both in the initial design of the primary sequence, and in the protocol used for synthesis of the secondary sequences. Horn et al1 have suggested a scheme for doing this, the overall design of which is shown therein.

The main recommendations for carrying out these syntheses are as follows:1, 2

Primary Sequence

For complex structures, the principal consideration when synthesising the primary target oligonucleotide is to combat the possible adverse effects on oligo yield due to the steric bulk of the oligo. This is done in two main ways: by using a large-pore CPG; and by introducing a spacer sequence to distance the branches from the CPG.

Increasing the pore size of the CPG to 2000Å or even 3000Å has been shown to greatly improve the quality of the synthesis. The steric constraints can be further reduced by using a lower nucleoside loading. A spacer sequence, typically T20, can be added between the primary sequence and inclusion of the branching molecules, and a further T2 spacer between the BMs themselves.

The coupling of the phosphoramidites, and subsequent levulinyl deprotection (90min for high BM content) and washing of the support is carried out as described above for simple primary sequences.

Secondary Sequence

Synthesis of the secondary sequences is best carried using a large excess of phosphoramidite reagents (10-fold excess with respect to each hydroxyl site) and a longer coupling time (e.g. 60s on an ABI 394 synthesiser). As in simpler structures (see above) the molar equivalents of phosphoramidites employed needs to be increased in accordance with the number of branched chains being extended.

The branched DNA can be detritylated, cleaved from the support and deprotected using ammonium hydroxide although conditions may need to be optimised. Note that for multiple branches the final detritylation may need to be increased.

References

  1. An improved divergent synthesis of comb-type branched oligodeoxyribonucleotides (bDNA) containing multiple secondary sequences, T. Horn, C-A. Chang and M.S. Urdea, Nucleic Acids Research, 25, 4835-4841, 1997.
  2. Chemical synthesis and characterization of branched oligodeoxynucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays, T. Horn, C-A. Chang and M.S. Urdea, Nucleic Acids Research, 25, 4842-4849, 1997.

 

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