Oligonucleotide synthesis routinely carried out from the 3' to 5' direction for no other reason than the ease of synthesis. However, a few applications required to synthesize oligonucleotides in the opposite sense. Using inverted base for reverse oligonucleotide synthesis is recommended for the following applications:
Formation of oligos containing hairpin loops with parallel strands
Oligonucleotides with hairpin loops are used for structural studies of duplex formation. Typically the strands of the stem of the hairpin are anti-parallel. However, by switching to using 5' inverted base for part of the synthesis of such an oligo, the strands of the hairpin stem will be in parallel orientation1. These parallel stranded oligos can be readily prepared with 5'--3' or 3'--5' sense.
Formation of nuclease resistant (5'-5', 3'-3') linkages
Designing antisense oligo-nucleotides for therapeutic applications have promoted considerable research activities on backbone modifications . Modifying the terminal linkages from the natural 3'-5' to 3'-3' and/or 5'-5' is a very efficient method to stabilize oligonucleotides against enzymatic degradation. Inverted linkages protect oligonucleotides against exonuclease activity, especially 3'-exonuclease activity which is by far the most significant enzymatic degradation route. Moreover, once degradation has occurred, the products are normal nucleosides with no toxicity concerns. Although this strategy has been applied successfully2 to the protection of internal linkages using alternating a,b nucleosides to maintain effective hybridization, the most simple strategy is to modify only the linkage at the 3' terminus3. This special kind of modification is useful for antisense assays as these oligonucleotides are more stable in living cells than normal DNAs.
Bio-Synthesis offers other related oligonucleotide modifications where synthesis in the opposite sense may be of interest would be:
- The preparation of oligos containing a base at the 3' terminus which is unsuitable for attachment to CPG, e.g., 2',3'-ddT and ddI .
- The synthesis of oligonucleotides on a support using a non-hydrolyzable linkage such that the 3' terminus is then available for polymerase extension.
Reference:
- J.H.v.d. Sande, N.B. Ramsing, M.W. Germann, W. Elhorst, B.W. Kalisch, E.v. Kitzing, R.T. Pon, R.C. Clegg, and T.M. Jovin, Science, 1988, 241, 551-557
- M. Koga, M.F. Moore, and S.L. Beaucage, J. Org. Chem., 1991, 56, 3757 .
- J.F.R. Ortigao, H. Rosch, H. Selter, A. Frohlich, A. Lorenz, M. Montenarh, and H. Seliger, Antisense Res. & Dev., 1992, 2, 129-146.
Custom Inverted Base Modified Oligonucleotide Services
There are many applications using reverse linked nucleotide modifications. They present nuclease resistance and permit the study of strand orientation effects on molecular biological function. When placing inverted base internally, the remaining portion of the oligo must be synthesized with reversed orientation. This type of special constructs must be quoted on a case-by-case basis.
Purification is recommended for inverted base modified oligos. Every oligo synthesized is strictly controlled for quality by using either MALDI-TOF mass spectrometry or polyacrylamide gel electrophoresis (PAGE) analysis. Final yields are determined using UV absorbance at OD260 In addition, we perform QC methods tailored to specific modifications, such as OD ratio measurement where appropriate. 08/13
3'-3' Inverted Base Modifications
3'-3' Inverted Base Oligonucleotide Modification:
3' Terminus
0.2 µmole $50
1.0 µmole $95
5.0 µmole $240
10 µmole $320
15 µmole $400
Internal
0.2 µmole $70
1.0 µmole $75
5.0 µmole $180
10 µmole $240
15 µmole $300
5'-5' Inverted Base Modifications
5'-5' Inverted Base Oligonucleotide Modification :
5' Terminus
0.2 µmole $70
1.0 µmole $75
5.0 µmole $180
10 µmole $240
15 µmole $300
Internal
0.2 µmole $70
1.0 µmole $75
5.0 µmole $180
10 µmole $240
15 µmole $300