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Nucleic Acids Research Advance Access published online on July 21, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn396
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine–pyrimidine junctions

Kenny Schlosser1, Jimmy Gu1, Jeffrey C. F. Lam1 and Yingfu Li1,2,*

1Department of Biochemistry and Biomedical Sciences and 2Department of Chemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

*To whom correspondence should be addressed. Tel: +9055259140, ext. 22462; Fax: +9055229033; Email: liying{at}mcmaster.ca

Received April 15, 2008. Revised June 5, 2008. Accepted June 5, 2008.

Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine–pyrimidine (pyr–pyr) junctions, which are cleaved 1–3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr–pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited kobs values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited ~1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.


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