1. R.P. Iyer, W. Egan, J.B. Regan, and S.L. Beaucage, J. Amer. Chem. Soc., 1990, 112, 1253-1254.
2. M. Overhoff and G. Sczakiel, EMBO Rep, 2005, 6, 1176-81.
3. J. Krutzfeldt, et al., Nature, 2005, 438, 685-9.
4. B.A. Kraynack and B.F. Baker, RNA, 2006, 12, 163-76.
5. B.J. Bergot and W. Egan, Journal of Chromatography, 1992, 599, 35-42.
A directed approach to the delivery of therapeutic oligonucleotides specifically to the liver has been to target the asialoglycoprotein receptor (ASGPR) using a suitable glycoconjugate. Indeed, ASGPR is the ideal target for delivery of therapeutic oligonucleotides to the liver since it combines tissue specificity, high expression levels and rapid internalization and turnover. The use of oligonucleotide glycoconjugates has led to significant advances in therapeutic delivery as evidenced by the work of Alnylam Pharmaceuticals and Ionis Pharmaceuticals using multivalent N-acetylgalactosamine (GalNAc) oligonucleotide conjugates.
Another paper3 describes a method for the inactivation of micro RNA (miRNA) that may help to elucidate their functions. It uses 2’-OMe-RNA oligonucleotides (23-mers, complementary to a target miRNA) with a cholesteryl group at the 3´terminus and phosphorothioates at positions 1 and 2 at the 5´end and at the last four positions at the 3´end. These oligos are called antagomirs. These molecules promote the cleavage of complementary miRNAs and thus should allow analysis of their function. The role of the PS linkages presumably is the stabilization against degradation in the mouse experiments as it is standard in the antisense field in such in vivo situations. And finally, a recent paper4 shows that PS does not systematically abolish siRNA activity, opening the way for some potentially less expensive stabilization of such molecules. Incorporation of 2’-OMe (in the sense strand) in combination with PS linkages should confer to siRNA increased resistance to degradation by nucleases, as well as prolonged serum retention. And it is also possible that such easy modification of siRNA may increase the specificity by eliminating sense strand recruitment in the RISC complex and thus reducing a source of off-target effect.
A new sulfurizing reagent must, therefore, exhibit all the good properties of Beaucage Reagent while adding good stability in solution on the synthesizer AND offering strong ability to sulfurize RNA linkages. We are happy to offer Sulfurizing Reagent II, 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT (2). Use of Sulfurizing Reagent II in RNA Synthesis
The authors note that the detailed studies of the molecular mechanisms of DNA repair pathways were made possible by using site-specifically modified oligonucleotides and that the availability of phosphoramidites to synthesize oligonucleotides with DNA lesions has contributed to the field. They illustrate the article using primarily structural studies in the following examples:
A wide range of common and alternative modifications in different synthesis scales and purification options are available at Gene Universal. By utilising a fully automated synthesis facility, controlled by a highly developed and proprietary production system, Gene Universal could ensure the high quality of our oligonucleotides.
The use of a sulfurizing reagent during the regular synthesis cycle using phosphoramidite chemistry has revolutionized the production of phosphorothioate oligonucleotide analogues. Undoubtedly, this ease of preparation of phosphorothioates has made this oligonucleotide modification by far the most common in research. Glen Research was one of the first sources of the sulfurizing reagent, 3H-1,2-benzodithiol-3-one 1,1-dioxide, popularly known as Beaucage Reagent (1).1 This sulfurizing reagent has found common use in the face of a plethora of rival reagents over the years because of its high efficiency, fast reaction time, and widespread availability. The one mild flaw we have found with Beaucage Reagent is that, although it is quite stable in acetonitrile solution in a silanized amber bottle, it is has relatively poor stability in solution once installed on the DNA synthesizer. Consequently, we have not been able to supply a sulfurizing solution, preferring to supply the powdered reagent along with an appropriate silanized bottle. The customer then weighs an appropriate amount of reagent into the silanized bottle and adds acetonitrile at a concentration of 1g/100mL. Over the years, we have considered other sulfurizing reagents but we were not able to find another reagent that exhibits the same fast sulfurization kinetics along with improved stability on the synthesizer. RNA Sulfurization
The simplest approach to MGB probe design is to use an MGB support, add a quencher molecule as the first addition and complete the synthesis with a 5'-fluorophore. Alternatively, a fluorophore support could be used with the 5' terminus containing a quencher molecule followed by a final MGB addition at the 5' terminus.
The most common usage for oligonucleotide phosphorothioates has been in the production of antisense oligodeoxynucleotides destined for use in identifying or modifying gene expression. Now, phosphorothioate linkages are popping up in the RNA world and sulfurizing RNA linkages with reagents like Beaucage Reagent has proved to be much more difficult than DNA linkages. The phosphorothioate (PS) linkage is a not-so-expensive way of increasing the stability of nucleic acids and increasing nuclease resistance of RNA. Now, it has been shown2 that fully PS oligos can promote the delivery of siRNA in cell culture. This siRNA uptake is sequence-independent and the length seems to vary between 30 and 70 nucleotides depending on the cell line. Even though this method is not yet as efficient as the cationic lipids, it opens the way to possible new methods. Reasons that may explain this are not understood at this time.
Phosphoramidites that allow the generation of oligonucleotides containing site-specific lesions have been vital components for studying the mechanism of DNA repair. New DNA lesions are still being discovered and the study of their biological consequences will require their site-specific incorporation into oligonucleotides. The authors conclude that the increased availability of phosphoramidites for the synthesis of lesion-containing oligonucleotides should facilitate many future discoveries in the broad area of DNA damage and repair.
Our experiments demonstrate that a 0.05 M solution of Sulfurizing Reagent II is recommended for the synthesis of RNA phosphorothioates. A sulfurizing time of 2-4 minutes generated oligophosphorothioates of high quality. This was true for both TOM-RNA and TBDMS-RNA monomers. As shown in Figure 2, Beaucage Reagent was significantly more sluggish than Sulfurizing Reagent II. Representative HPLC analyses5 of RNA oligos are shown in Figure 3. The chromatogram on the left was obtained from sulfurizing U-TOM-RNA linkages for 60 seconds with Beaucage Reagent. The large n-1 peak is due to incomplete stepwise sulfurization and accumulation of deletions. The chromatogram on the right was an identical synthesis except using Sulfurizing Reagent II. Individual RNA sequences, especially those containing stretches of purine nucleoside residues are more difficult to sulfurize irrespective of the reagent used. To obtain a high degree of sulfurization with those oligonucleotides, a 0.1 M solution of Sulfurizing Reagent II and/or extended contact time may be required.