Figure 1. (a) Ribonucleoside phosphoramidite building blocks for commercialized RNA synthesis methods using either 5′‐O‐DMT‐2′‐O‐silyl ( 1 and 2) or 5′‐O‐silyl‐2′‐O‐ACE chemistry ( 3); (b) A collection of commercially available modified nucleosides for solid‐phase synthesis of RNA by phosphoramidite chemistry. All building blocks are compatible with the 5′‐O‐DMT‐2′‐O‐silyl protection scheme. Base‐modified nucleosides 4– 36 are available with the 2′‐O‐TBDMS protecting group. The asterisk indicates additional availability as 2′‐O‐TOM‐protected phosphoramidite; # denotes availability via custom synthesis service using 2′‐O‐ACE chemistry (Dharmacon).

Figure 2. Selected examples of postsynthetic RNA modification strategies. (a) Thioether formation with α‐haloacetamides and disulfide formation with methanethiosulfonate reagents; (b) Palladium‐catalyzed crosscoupling of terminal alkynes to 5I‐U on the solid support (known as Sonogashira crosscoupling); (c) Amino groups reacting with isocyanate or isothiocyanate form urea or thiourea bonds, reactions with NHS or STP esters give amide bonds. R groups are biophysical labels or reporter groups such as fluorophores, ion complexation reagents, photocrosslinking reagents, or spin labels.

Figure 3. Strategies for enzymatic ligation of RNA fragments. (a) 5′‐Phosphorylated donor RNA and 3′‐hydroxyl‐terminated acceptor RNA are aligned by a splint oligonucleotide for ligation with T4 DNA ligase in the presence of ATP; (b) 5′‐Phosphorylated donor and 3′‐hydroxyl‐terminated acceptor oligonucleotides hybridize and preorganize the fragments for ligation with T4 RNA ligase in the presence of ATP; (c) T4 RNA ligase can join an activated (adenylated) donor fragment to a 5′‐phosphorylated acceptor fragment in the absence of ATP; (d) DNA‐catalyzed ligation of triphosphorylated donor to 3′‐hydroxyl‐terminated acceptor.