Solid-phase synthesis of oligoribonucleotides using T4 RNA ligase and T4 polynucleotide kinase

Abstract

The concept of solid-phase synthesis of oligoribonucleotide using T4 RNA l ipase and T4 polynucleotide kinase has been proposed and tested with model homo-oligoribonucleotides. The method consists of the immobilization of the first oligomer block at the 3′-terminus on a solid support followed by a chain elongation in the 5′-direction with trinucleoside diphosphates using T4 RNA-ligase and phosphorylation using polynucleotide kinase. Hydrazides of Biogel P-300, Sepharose 4B and cellulose were tested as solid supports for immobilization of initial oligomers. The properties of supports were rated on reactivities of immobilized 5′-phosphorylated oligomers as phosphate donors in the solid phase reactions, hydrodynamical properties and capacity to eliminate donor molecules spontaneously during reactions. Hydrazide of Sepharose 4B appeared to be a more suitable support because of better hydrodynamic properties and highest reactivities of immobilized donors. Saturated concentrations of RNA ligase and polynucleotide kinase and optimal time of joining reaction were determined. In a model experiment ApApA was twice attached to the immobilized hydrazide of Sepharose 4B donor (pA) 6pA ox. The yield of (Ap) 12 was 25%.