FIG. 1.

Preparation of ³²P-labeled site-specific photo-cross-linking probes. The double-stranded probes were prepared by using long (∼80 nucleotides) synthetic template oligonucleotides (shown in the lower part), chemically derivatized oligonucleotides labeled at the 5′ end with ³²P, and appropriate upstream primers. The “R” stands for an azidophenacyl moiety coupled to a phosphorothioate residue in the downstream primer, and the asterisk indicates a ³²P radiolabel. For details, see Materials and Methods. The lower part shows the actual sequences of the long template oligonucleotides used to generate the probes. Note that the U1 and U6 PSEA template oligonucleotides have exactly the same sequence with the exception of five base changes (denoted by underlines) within the sequence of the PSEA (shown in boldface). The mutant template contains seven base changes at the upstream end of the U1 PSEA which are indicated by lowercase letters.