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In vitro selection of nucleoprotein enzymes

Nature Biotechnology volume 19, pages 650–655 (2001)Cite this article

Abstract

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.

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Figure 1: L1 ligase, L1-N50 pool, and selection scheme.
Figure 2: Progress of the L1-N50 selections.
Figure 3: Protein-dependent ribozyme sequences and structures.
Figure 4: Ribozyme activity with inactivated protein samples.
Figure 5: Aptamer competition assays.
Figure 6: Binding and ligation activity as a function of protein concentration.

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Acknowledgements

We would like to thank Jay Hesselberth and Christopher Myers for providing purified Cyt18 protein, Jamie Bacher for providing E. coli TrpRS, and Colin Cox for supplying unpublished aptamer sequences. This work was supported by DARPA contract no. N65236-98-1-5413.

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  1. Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, 78712, TX

    Michael P. Robertson & Andrew D. Ellington

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Robertson, M., Ellington, A. In vitro selection of nucleoprotein enzymes. Nat Biotechnol 19, 650–655 (2001). https://doi.org/10.1038/90256

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