| 1. |
- Semenyuk, Andrey, et al.
(författare)
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A base-stable dithiomethyl linker for solid-phase synthesis of oligonucleotides
- 2007
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Ingår i: Tetrahedron Letters. - : Elsevier BV. - 0040-4039 .- 1359-8562. ; 48:3, s. 469-472
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Tidskriftsartikel (refereegranskat)abstract
- A novel linkage, useful for the synthesis of oligonucleotides is described. The linking function is compatible with all conditions used for oligonucleotide synthesis, orthogonal to all other protecting groups, but regenerates 3′-OH rapidly upon mild reduction under aqueous conditions. This method is employed in the removal of depurinated fragments during the synthesis of oligonucleotides.
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| 2. |
- Semenyuk, Andrey, et al.
(författare)
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Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays
- 2006
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 356:1, s. 132-141
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Tidskriftsartikel (refereegranskat)abstract
- A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.
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| 3. |
- Semenyuk, Andrey, 1978-
(författare)
-
Novel Methods for Synthesis of High Quality Oligonucleotides
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC.In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties.The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.
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| 4. |
- Semenyuk, Andrey, et al.
(författare)
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Synthesis of RNA Using 2'-O-DTM Protection
- 2006
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 128:38, s. 12356-12357
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Tidskriftsartikel (refereegranskat)abstract
- tert-Butyldithiomethyl (DTM), a novel hydroxyl protecting group, cleavable under reductive conditions, was developed and applied for the protection of 2′-OH during solid-phase RNA synthesis. This function is compatible with all standard protecting groups used in oligonucleotide synthesis, and allows for fast and high-yield synthesis of RNA. Oligonucleotides containing the 2′-O-DTM groups can be easily deprotected under the mildest possible aqueous and homogeneous conditions. The preserved 5′-O-DMTr function can be used for high-throughput cartridge RNA purification.
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| 5. |
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