Isolation of oligoribonucleotides containing intramolecular cross-links.

8. Pearson, R. L., Weiss, J. F., and Kelmers, A. D. (1971) Biochim. of the other sample. We report here a systematic Biophys. Acta 228, 770–774. study of this interesting phenomenon which sug9. Christensen, L., et al. (1995) J. Pept. Sci. 3, 175–183. gests DPAGE as a general method for the analysis 10. Noble, S. A., et al. (1995) Drug. Dev. Res. 34, 184–185. and isolation of intramolecularly cross-linked oligo11. Cubellis, M. V., Marino, G., Mayol, L., Picialli, G., and Sannia, ribonucleotides. G. (1985) J. Chromatogr. 329, 406–414. The two cross-linked ribozymes, ribozyme A (Rz A) and ribozyme B (Rz B), were prepared and purified as previously described (1). Rz A and B as well as the corresponding non-cross-linked ribozymes, obtained by the reduction of the cross-linked samples with Isolation of Oligoribonucleotides Containing dithiothreitol (1), were subjected to DPAGE conIntramolecular Cross-Links taining 8, 12, 16, 20, and 24% acrylamide (0.04 1 20 1 40-cm gel; acrylamide:bisacrylamide 19:1). The Snorri Th. Sigurdsson and Fritz Eckstein distance that the cross-linked and non-cross-linked Max-Planck-Institut für Experimentelle Medizin, material migrated in the gels was subsequently meaHermann-Rein-Strasse 3, D-37075 Göttingen, Germany sured. To facilitate a comparison of results from all the gel analyses, the mobilities of Rz A and B were normalized, assuming that the non-cross-linked Received January 3, 1996 samples had migrated 30 cm in all the gels. The results are presented in the simulated gel shown in Recently we developed a cross-linking methodolFig. 2. ogy for probing the tertiary structure of RNA and Inspection of Fig. 2 reveals an enormous difference used it to assess the catalytic competence of two difin the relative mobility of Rz A to the non-crossferent three-dimensional models of the hammerhead linked material, from 22.7 cm in a 24% gel to 32.9 ribozyme (1). In essence, our strategy was to introcm in an 8% gel, a difference of 10.2 cm. The same duce conformational constraints into two ribozyme effect is also observed for Rz B, although the differconstructs by the means of a disulfide cross-link beence in relative mobility is less than for Rz A. To tween helices I and II and to use the catalytic effiour knowledge, there is only one other report in the ciencies of these two cross-linked ribozymes for evalliterature that describes this phenomenon; Grabowuation of the structural models. We prepared two ski et al. (5) analyzed a ca. 230-nucleotide-long ‘‘larribozymes (Fig. 1), each carrying two 2 *-amino-modiiat’’ intermediate in messenger RNA splicing by fications which were reacted with an aromatic isoDPAGE and found that the apparent length was dethiocyanate containing a protected mercaptan funcpendent upon the percentage acrylamide used and tionality. Deprotection of the thiols, followed by that it migrated as a longer oligomer under all condiair-oxidation yielded a mixture of cross-linked and tions tested (4–10% acrylamide). By the same token, non-cross-linked material. Since the catalytic efficiencies of the cross-linked ribozymes could be affected by contamination of non-cross-linked material we needed to completely separate the two species. Denaturing polyacrylamide gel electrophoresis (DPAGE) has previously been used for the isolation of intramolecular cross-links (2–4) in spite of the fact that they have the same nucleotide sequence, the same charge, and nearly the same mass as the corresponding non-cross-linked material. Surprisingly, in a preliminary attempt to isolate the two cross-linked ribozymes by 20% DPAGE we could only separate one adequately from the non-cross-linked material. However, we found that the relative mobility of cross-linked to non-cross-linked material varied greatly with the percentage of acrylamide in the denaturing gels and this facilitated the separation FIG. 1. The sequence and secondary structure of the two hammerhead ribozyme constructs used in this study, shown here without the substrate. Each ribozyme contained two 2*-amino-groups that 1 To whom correspondence should be addressed. Fax: (/49)-5513899-388. E-mail: [email protected]. were subsequently modified and cross-linked. The position of the cross-link in Rz A and Rz B is indicated by black and gray lines, 2 Abbreviation used: DPAGE, denaturing polyacrylamide gel electrophoresis. respectively.