Figure 3.
Synthesizing pyrene-labeled P4–P6 by derivatization-ligation (path 1) or by derivatization-annealing (path 2). (A) A 15-mer or 24-mer oligoribonucleotide incorporating a single 2′-amino or 2′-(2-aminoethoxy) nucleotide is derivatized with pyrene as in Figure 1C and purified by PAGE. (B) To complete path 1, the 15-nt product from the derivatization step is ligated to the remaining 145 nt of P4–P6 using a DNA splint and T4 DNA ligase; the product is purified by PAGE. (C) To complete path 2, the 24-nt product from the derivatization step is annealed to the remaining 136 nt of P4–P6 without covalent ligation.