Production of polymers from nucleotide and/or non-nucleotide units using RNA ligase, useful for

摘要

An RNA ligase is used in a process for producing polymers from nucleotide and/or non-nucleotide units by coupling in the 3' to 5' direction. Independent claims are also included for the following: (1) a process for producing natural or modified polymers with a defined sequence of nucleotide and/or non-nucleotide units comprises: (a) ligating a donor of formula (I) or (II) to a 3'-unprotected acceptor of formula (III) or (IV) using an RNA ligase in the presence of divalent metal cations; (b) deprotecting the product by: (i) cleaving the (rM)n protecting group of (III) with a ribonuclease (RNase) or by alkaline hydrolysis; or (ii) cleaving the (rM)ndU protecting group of (IV) with a uracil DNA glycosylase and an apurinic endonuclease or by alkaline hydrolysis; (c) transferring a terminal group p to the 5'-hydroxy group of the ligation product using a polynucleotide kinase; (d) optionally repeating steps (a)-(c); and (e) optionally purifying the product: (2) a process for producing natural or modified polymers with a defined sequence of nucleotide and/or non-nucleotide units, comprising: (a) ligating a donor of formula (V) or (VI) to a 3'-unprotected acceptor of formula (VII) or (VIII) using an RNA ligase in the presence of divalent metal cations; (b) deprotecting the product by: (i) cleaving the p(rM)n protecting group of (VII) with a RNase or by alkaline hydrolysis; or (ii) cleaving the p(rM)ndU protecting group of (VIII) with a uracil DNA glycosylase and an apurinic endonuclease or by alkaline hydrolysis; (c) optionally repeating steps (a) and (b); and (d) optionally purifying the product; and (3) acceptor molecules of formula (III), (IV), (VII) and (VIII). (I) p(dNp)m; (II) p(rNp)m; (III) (rM)ndN; (IV) (rM)ndU-rN; (V) (dN)m; (VI) (rN)m; (VII) p(rM)ndNp; (VIII) p(rM)ndU-rNp; p = a phosphate, phosphorothioate, phosphonate (preferably methylphosphonate or H-phosphonate), phosphoramidate, formacetal, phosphorodithioate, boranophosphate or phosphotriester group; dN = a natural or base- or sugar-modified deoxyribonucleoside or a ligatable non-nucleoside unit; rN = a natural or base- or sugar-modified ribonucleoside or a ligatable non-nucleoside unit; m = 1 or more; rM = a purine or pyrimidine ribonucleoside; n = 1 or more; dU = deoxyuridine.