The 5′-triphosphates are the building blocks for the enzymatic synthesis of DNAs and RNAs. This unit presents a protocol for the convenient synthesis of 2′-deoxyribo- and ribo-nucleoside 5′-triphosphates (dNTPs and NTPs) containing all the natural bases and the modified bases. This one-pot synthesis can also be applied to prepare the triphosphate analogs that contain sulfur or selenium atoms replacing the non-bridging oxygen atoms of the alpha phosphates of the triphosphates. These S- or Se-modified dNTPs and NTPs can be used to prepare diastereomerically-pure phosphorothioate nucleic acids (PS-NAs) or phosphoroselenoate nucleic acids (PSe-NAs, i.e., one type of selenium-derivatized nucleic acids: SeNA). Even without extensive purification, the synthesized dNTPs or NTPs are of high quality and can be directly used in DNA polymerization or RNA transcription. Synthesis and purification of the 5′-triphosphates, analysis and confirmation of natural and sulfur-or selenium-modified nucleic acids are described in this protocol unit.
Modified deoxy-and ribo-nucleoside triphosphates are chemically synthesized in multiple steps due to the protection and deprotection of the nucleoside functionalities. To conveniently synthesize the S-modified triphosphates for enzymatically preparing phosphorothioate DNAs and RNAs (PS-DNA and PS-RNA) as potential therapeutics, herein we report a one-pot strategy to synthesize the deoxy-and ribo-nucleoside 5′-(α-P-thio)triphosphates (dNTPαS and NTPαS) without protecting any nucleoside functionalities. This facile synthesis is achieved by treating the nucleosides with a mild phosphitylating reagent, reacting selectively with the 5′-hydroxyl group of each unprotected nucleoside, followed by sulfurization and hydrolysis to afford the crude dNTPαS and NTPαS analogs (mixtures of S p and R p diastereomers). We also demonstrated that after just simple precipitation (without HPLC and ion-exchange purification), the quality of the synthesized dNTPαS and NTPαS analogs is excellent for direct DNA polymerization and RNA transcription, respectively. Since Klenow DNA polymerase and T7 RNA polymerase accept the S p diastereomers of dNTPαS and NTPαS analogs, respectively, while the R p diastereomers are neither substrates nor inhibitors, the diastereomerically-pure PS-DNAs and PS-RNAs can be conveniently synthesized enzymatically.sulfur modification, S-derivatized nucleic acid, nucleoside triphosphates, nucleoside phosphorothioates, phosphorothioate DNA and RNA, diastereomers, DNA and RNA polymerases
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