Recombinant and In Vitro RNA Synthesis

N. Ruth Zearfoss, Sean P. Ryder, Zearfoss, N. Ruth, Ryder, Sean P.

Many experimental strategies for determining nucleic acid function require labeling the nucleic acid with radioisotopes or a chemical tag. Labels enable nucleic acid detection, yield information about its state, and can serve as a handle by which the nucleic acid and associated factors can be purified from a mixture. Radioactive phosphate is commonly added to the 5' or 3' end of an oligonucleotide post synthesis using enzyme-catalyzed reactions. In contrast, chemical tags are usually added during synthesis or using reactive groups that are incorporated during synthesis. Here, we present protocols for post-synthetic conjugation of chemical tags to unmodified RNA or DNA oligonucleotides. The approach can be used to attach fluorescent dyes and biotin groups to oligonucleotides and to immobilize oligonucleotides to a solid support. Oligonucleotides tagged with fluorescent dyes are readily detected in both gel- and plate reader-based assays, while biotin- or resin-conjugated oligonucleotides are useful tools for affinity purification. Fluorescent end-labeling provides several advantages over radioactive labeling, reducing radioactivity-associated hazards and yielding a labeled molecule that does not decay while providing the sensitivity required for many procedures.