| Technical Resources |
| Dharmacon 2'-ACE RNA
Chemistry |
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Dharmacon RNA synthesis chemistry is based on a novel protecting group scheme.1
A new class of silyl ethers is used to protect the 5´-hydroxyl (5´-SIL) in
combination with an acid-labile orthoester protecting group on the 2´-hydroxyl
(2´-ACE).2 This set of protecting groups is then used with standard
phosphoramidite solid-phase synthesis technology.3 The structures of the
protected and functionalized ribonucleoside phosphoramidites currently in use
are as illustrated in Figure 1.
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Figure 1 Protected RNA nucleoside phosphoramidites for
Dharmacon 2´-ACE RNA synthesis chemistry |
| RNA oligonucleotides are synthesized in a stepwise
fashion using the nucleotide addition reaction cycle illustrated in Figure 2.
Each nucleotide is added sequentially (3´- to 5´-direction) to a solid
support-bound oligonucleotide. The first nucleoside at the 3´-end of the chain
is covalently attached to a solid support. The nucleotide precursor, a
ribonucleoside phosphoramidite, and activator are added (step i in Figure 2),
coupling the second base onto the 5´-end of the first nucleoside. The support
is washed and any unreacted 5´-hydroxyl groups are capped with acetic anhydride
to yield 5´-acetyl moieties (step ii). The P(III) linkage is then oxidized to
the more stable and ultimately desired P(V) linkage (step iii). At the end of
the nucleotide addition cycle, the 5´-silyl group is cleaved with fluoride
(step iv). The cycle is repeated for each subsequent nucleotide. |
Figure 2 Outline of Dharmacon RNA Synthesis Cycle |
(i) couple next nucleoside with S-ethyl-tetrazole
catalyst, 60 seconds
(ii) cap unreacted 5´-hydroxyls, 20 seconds
(iii) oxidize phosphorus linkage
(iv) 5´-deprotection with triethylammonium fluoride ions (TEAHF), 30 seconds |
Following synthesis, the methyl protecting groups on
the phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S2Na2) in DMF.4
The deprotection solution is washed from the solid support bound
oligonucleotide using water. The support is then treated with 40% methylamine5
in water for 10 minutes at 55 °C. This releases the RNA oligonucleotides into
solution, deprotects the exocyclic amines and modifies the 2´-ACE groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this stage.
The 2´-orthoester groups are the last protecting groups to be removed. The
structure of the 2´-ACE protected RNA immediately prior to 2´-deprotection is
as represented in Figure 3. |
Figure 3 Structure of 2´-ACE protected RNA immediately prior
to 2´-deprotection. |
The ethylene glycol monoacetate orthoester group
(Figure 1) that was developed by Dharmacon Research has the following
innovative properties. It is stable to the conditions of nucleoside
phosphoramidite synthesis and oligonucleotide synthesis. However, after
oligonucleotide synthesis the oligonucleotide is treated with methylamine which
not only cleaves the oligo from the solid support but also removes the acetyl
groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the
orthoester are less electron withdrawing than the acetylated precursor. As a
result, the modified orthoester becomes more labile to acid-catalyzed
hydrolysis (refer to mechanism in Figure 4). Specifically, the rate of cleavage
is approximately 10 times faster after the acetyl groups are removed.
Therefore, this orthoester possesses sufficient stability in order to be
compatible with oligonucleotide synthesis and yet, when subsequently modified,
permits deprotection to be carried out under relatively mild aqueous conditions
compatible with the final RNA product.
The hydrophilic character of the 2´-protecting groups predicts that
2´-protected RNA of any length or sequence will be readily soluble in water.
Base composition and length have been found to have no effect on the solubility
of the 2´-protected RNA oligos. To date, all lengths and sequences synthesized
with 2´-ACE chemistry have been water soluble. This enables the routine
handling of 2´-protected RNA in water.
When ready to remove the 2´-protecting groups, the orthoesters are readily
hydrolyzed under acid catalysis by the mechanism in Figure 4.6
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Figure 4 Generalized mechanism for acid catalyzed hydrolysis
of 2´-orthoesters. |
As the 2´-protecting groups are very hydrophilic and
readily solvated by water, acid-catalyzed hydrolysis proceeds to completion
regardless of sequence or length.
5´-SIL-2´-ACE oligonucleotide chemistry is a definitive advance in RNA
synthesis technology. Nucleoside coupling yields are comparable to DNA and are
routinely <60 seconds. The final acid deprotection is mild, fast and
amenable to subsequent use with minimal handling. Analysis and purification via
PAGE or HPLC are possible with any sequence while in the stable 2´-protected
form. This property minimizes opportunities to degrade the RNA, while also
making it possible to analyze troublesome sequences with strong secondary
structure. 5´-SIL-2´-ACE chemistry has enabled the routine synthesis of RNA
oligonucleotides of unprecedented quality. Studies to expand the range of
products and modifications are in progress.
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| Bibliography |
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(a) Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996. (b) Scaringe,
S. A. and Caruthers, M. H. "Silyl Ether Protection of the 5´-Hydroxyl during
Solid Phase Oligonucleotide Synthesis," in preparation. (c) Scaringe, S. A. and
Caruthers, M. H. "5´-O-Silyl Ethers in Conjunction with Acid-labile
2´-O-orthoesters for the Solid Phase Synthesis of RNA." in preparation.
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Scaringe, S. A., Wincott, F. E. and Caruthers, M. H. "Novel RNA Synthesis
Method Using 5´-Silyl-2´-Orthoester Protecting Groups," J. Am. Chem. Soc., 120,
11820-11821 (1998).
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(a) Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc. 103, 3185-3191
(1981). (b) Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett. 22,
1859-1862 (1981).
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Dahl, B. J., Bjergarde, K., Henriksen, L. and Dahl, O., Acta Chem. Scand. 44,
639-641(1990).
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(a) Reddy, M. P., Hanna, N. B. and Farooqui, F. Tetrahedrom Lett., 25,
4311-4314 (1994). (b) Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz,
D.; Workman, C.; Sweedler, D.; Gonzalez, C.; Scaringe, S. and Usman, N.,
Nucleic Acids Res. 1995, 23, 2677-2684.
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(a) Griffin, B. E., Jarman, M., Reese, C. B. and Sulston, J. E. Tetrahedron 23,
2301-2313 (1967). (b) Griffin, B. E., Jarman, M., Reese, C. B. and Sulston, J.
E. Tetrahedron 23, 2315-2331 (1967).
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