Solid-phase phosphoramidite-synthesis of long oligonucleotide molecules

A method of solid-phase phosphoramidite-synthesis of an oligonucleotide molecule comprising between 50 and 500 nucleotides, said method comprising the following steps:

• a) providing a blueprint for a desired oligonucleotide sequence;
• b) providing a solid support functionalized with a linker moiety and providing a plurality of ribonucleotide monomer subunits and modified ribonucleotide monomer subunits, locked nucleic acids and non-nucleosidic monomers, wherein each of the monomer subunits comprises a protected hydroxyl or specifically protected 5′-hydroxyl group;
• c) treating the solid support for a predetermined time with an acidic deblocking solution to expose free 5′-hydroxyl groups;
• d) washing the solid support with an appropriate solvent to remove the deblocking solution;
• and the cleaved protecting group;
• e) treating the solid support with a coupling mixture comprising an activating reagent and ribonucleotide monomer subunits, each comprising a phosphoramidite group and a blocked hydroxyl group to obtain phosphite triester linked ribonucleotide monomer subunits;
• f) washing the solid support with an appropriate solvent to remove the coupling mixture;
• g) oxidizing, sulfurizing, or thiolating the phosphite triester linked ribonucleotide monomer subunits to provide phosphate triester or thiophosphate triester linked ribonucleotide monomer subunits, whereby the oxidation or thiolation is carried out with an appropriate reagent;
• h) washing the solid support with an appropriate solvent to remove the oxidizing or thiolating solutions;
• i) treating the phosphate triester or thiophosphate triester linked ribonucleotide monomer subunits with a mixture of one or more capping reagents to block any uncoupled support-bound 5′ hydroxy byproduct, whereby the mixture of one or more capping reagents has less than 15equivalents of capping reagents based on the loading of the solid phase;
• j) washing the solid support with an appropriate solvent to remove the capping solutions;
• k) repeating steps c) to j) until the desired amount of ribonucleotide units is coupled to the solid support to obtain the desired oligonucleotide molecule;
• l) treating the solid support with a solution of an alkyl amine, a cyclic or a bicyclic amine in an appropriate solvent to remove phosphorous protecting groups;
• m) washing the solid support with an appropriate solvent to remove the phosphorous deprotection reagent;
• n) deprotecting and cleaving the oligoribonucleotide compound from the solid support to obtain the crude oligonucleotide molecule, and
• o) optionally, purifying the crude oligonucleotide molecule.