Figure 5.

Strategies for juxtaposing reactive ends for enzymatic ligation. Pre-orientation of 5′-p and 3′-OH of the RNA substrate is achieved (a) by hybridization to a splint. This strategy can be used for ligation with T4 DNA ligase (DNA splint required), T4 RNA ligase 1 (RNA splint required) and T4 RNA ligase 2 (DNA or RNA splint) (68); (b) by hairpin and linear helper oligonucleotides (87); (c) by a gap splint that upon hybridization leaves the terminal two or three nucleotides of both ends single stranded at the ligation junction (88); (d) by favorable intrinsic structures (i.e. dumbbell folds (17)). Strategies (b), (d) and (c) are favorable for ligation with T4 RNA ligase 1. All strategies may be used also for chemical RNA ligation. Note that in this case a 3′-phosphate and a 5′-OH group are favorable.