New methods for RNA synthesis

RNA synthesis is less efficient than DNA synthesis, owing the problems caused by the presence of the 2'-hydroxyl group. Protection and deprotection of this group during solid-phase synthesis reduces coupling efficiency and leads to side-reactions, and constructs longer than 50 nucleotides in length (far smaller than important biologically active RNA molecules) are difficult to prepare. Alternative methods including transcription and enzymatic ligation prevent the site-specific incorporation of multiple modifications at sugars, bases and phosphates and are limited in scale.

Scheme showing the use of click chemistry in the synthesis of the hammerhead ribozyme We have developed a method which we call “click RNA ligation” (View paper) that overcomes the above limitations and allows the synthesis of RNA molecules and DNA/RNA chimeras up to 100 nucleotides in length. In this we synthesize individual RNA oligonucleotides by automated solid phase synthesis, and use the CuAAC reaction to ligate chemically modified RNA strands. This reaction is efficient, orthogonal to functional groups present in nucleic acids and works well in aqueous media. We have used click RNA ligation to synthesize catalytically-active variants of the hairpin ribozyme and the hammerhead ribozyme.

This chemistry could be applied to the synthesis of many important RNA molecules such as riboswitches, siRNA delivery systems, multivalent aptamers and components of ribosomes.