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Nucleic Acids Research, 1992, Vol. 20, No. 9 2223-2232
© 1992

MOLECULAR BIOLOGY

Effect of the higher-order structure of tRNAs on the stability of hybrids with oligodeoxyribonucleotides: separation of tRNA by an efficient solution hybridization

Yoshinori Kumazawa+, Takashi Yokogawa, Hiromichi Tsurui1, Kin-ichiro Miura2 and Kimitsuna Watanabe*

Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology 4259 Nagatsuta-machi, Midori-ku, Yokohama 227, Japan 1Department of Pathology, Juntendo University School of Medicine Hongo, Bunkyo-ku, Tokyo 113, Japan 2Department of Industrial Chemistry, Faculty of Engineering, the University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

*To whom correspondence should be addressed

Received February 26, 1992. Accepted March 30, 1992.

In the course of developing a method to purify a single tRNA species efficiently, we have examined hybridization effciencies between some tRNAs and short oligodeoxyribonucleotide probes both by the filter and solution hybridization methods without denaturants. The hybridization efficiencies varied considerably among probes which are complementary to different regions of the tRNAs, although there was little efficiency variation in the probes toward DNA substrates including the same nucleotide sequence. This efficiency variation was shown to be due to tRNA specific higher-order structures as well as a hypermodified nucleotide in the anticodon loop. Characterization of the tRNA-probe hybrids by both nondenaturing gel electrophoresis and chemical modification showed the existence of two stable hybridizing states as a function of ionic strength. Our results indicate that RNA molecules with a number of intramolecular base pairings are able to form stable hybrids with complementary sequences under nondenaturing conditions. On the basis of these data, an appropriate probe was designed to successfully purify yeast tRNAPhe by making a tRNAPhe-probe hybrid, which has a longer retention time in hydroxyapatite high performance liquid chromato graphy than the tRNAPhe itself.

+Present address: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA


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