Ronald Micura

Ribonucleic acids (RNAs) play a vital role in living organisms. Many of their cellular functions depend critically on chemical modification. Methods to modify RNA in a controlled manner—both in vitro and in vivo — are thus essential to evaluate and understand RNA biology at the molecular and mechanistic levels. The diversity of modifications, combined with the size and uniformity of RNA (made up of only 4 nucleotides) makes its site‐specific modification a challenging task that needs to be addressed by complementary approaches. One such approach is solid‐phase RNA synthesis. We discuss recent developments in this field, starting with new protection concepts in the ongoing effort to overcome current size limitations. We continue with selected modifications that have posed significant challenges for their incorporation into RNA. These include deazapurine bases required for atomic mutagenesis to elucidate mechanistic aspects of catalytic RNAs, and RNA containing xanthosine, N 4 ‐acetylcytidine, 5‐hydroxymethylcytidine, 3‐methylcytidine, 2′‐OCF 3 , and 2′‐N 3 ribose modifications. We also discuss the all‐chemical synthesis of 5′‐capped mRNAs and the enzymatic ligation of chemically synthesized oligoribonucleotides to obtain long RNA with multiple distinct modifications, such as those needed for single‐molecule FRET studies. Finally, we highlight promising developments in RNA‐catalyzed RNA modification using cofactors that transfer bioorthogonal functionalities.