Abstract: Cleavable nucleotide analogs are provided. The nucleotide analog includes a nucleotide molecule attached to a cleavable moiety wherein the cleavable moiety comprises a protective group and/or a linker attached to a fluorophore. The cleavable moiety is linked to the oxygen atom of the 3?-OH of the pentose of the nucleotide molecule. The nucleotide analogs can be used in making polynucleotide molecules using template independent polymerases. The nucleotide analogs can act as reversible terminators during DNA sequencing by synthesis. The cleavage of the cleavable moiety restores a free 3?-OH functional group allowing growth of the polynucleotide molecule. The general structures as well as proposed synthetic schemes for the nucleotide analogs are also provided.

Abstract: Described herein are methods for the controlled de novo synthesis of RNA oligonucleotides using enzymatic catalysis. For example, provided herein are methods for preparing RNA oligonucleotides via controlled, template-independent addition of nucleotides to an initiator oligonucleotide 3?-terminus via enzymatic catalysis (also known as terminal transferase activity). Single nucleotides can be iteratively added by a compatible polymerase (e.g., a poly(N) polymerase such as a poly(U) polymerase) until a desired RNA oligonucleotide sequence is synthesized. Also provided are nucleotides and polymerases useful in the methods described herein.