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Issue 7, 2011
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Characterization of non-8–17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

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Abstract

RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8–17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8–17 sequences obtained from a previous in vitro selection experiment wherein the 8–17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (kobs from 0.003 to 1 min−1), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.

Graphical abstract: Characterization of non-8–17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

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Publication details

The article was received on 26 Jan 2011, accepted on 06 Apr 2011 and first published on 27 Apr 2011


Article type: Paper
DOI: 10.1039/C1MB05034F
Citation: Mol. BioSyst., 2011,7, 2139-2146
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    Characterization of non-8–17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

    J. C. F. Lam, S. O. Kwan and Y. Li, Mol. BioSyst., 2011, 7, 2139
    DOI: 10.1039/C1MB05034F

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