Fig. 1: General overview of the controlled, template-independent enzymatic RNA oligonucleotide synthesis process.
From: Template-independent enzymatic synthesis of RNA oligonucleotides
a, Three primary components are required for carrying out an enzymatic extension: 3′-blocked reversible terminator nucleotides, enzymes capable of their robust and indiscriminate incorporation, and an initiator oligonucleotide. The reversible terminator group stops uncontrolled polymerization by the enzyme and limits extension to a single incorporation event. The initiator oligonucleotide may vary in terms of sequence and length. It can also be bound to a solid support or feature other modifications such as a 5′-fluorophore or functional handle. b, A typical cycle of enzymatic synthesis begins with (1) extension of the initiator oligonucleotide in the presence of an RT-NTP and enzyme. A deblocking step (2) then occurs to remove the reversible terminator group from the extended oligonucleotide, allowing the next cycle of synthesis to commence. When the desired length and composition have been reached, the final oligonucleotide product is isolated.
