FIG 2.

Ligation of nicked duplexes with paired and mispaired 5′-PO₄ ends. (A) The structure of the nicked duplex substrate is shown at the top, with the 5′ ³²P label at the nick denoted by a closed circle. The 3′-OH strand is all RNA. The 5′ N-X base pair at the nick (highlighted in the shaded box) is variable. Ligase reactions were performed as described in Materials and Methods. The yields of the sealed 36-mer RNApDNA product are plotted as a function of time; the experiments are presented in sets of four according to the identity of the X nucleobase, with the N nucleobase (specified to the right of each curve) being the variable within the set. Each datum is the average of at least 3 separate experiments ± SEM. (B) The initial rates of sealing of the indicated 5′ N-X nicks were calculated by linear regression in Prism.