Nucleic Acids Research Advance Access originally published online on April 8, 2009
Nucleic Acids Research 2009 37(11):3545-3557; doi:10.1093/nar/gkp222
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Nucleic Acids Research, 2009, Vol. 37, No. 11 3545-3557
© 2009 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.
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A genotype-to-phenotype map of in vitro selected RNA-cleaving DNAzymes: implications for accessing the target phenotype
1Department of Biochemistry and Biomedical Sciences and 2Department of Chemistry, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
*To whom correspondence should be addressed. Tel: +1 905 525 9140 ext. 22462; Fax: +1 905 522 9033; Email: liying{at}mcmaster.ca
Received February 5, 2009. Revised March 18, 2009. Accepted March 23, 2009.
Herein, we describe a case study into the population dynamics of in vitro selection, using RNA-cleaving DNAzymes as a model system. We sought to understand how the composition of the population can change over time in response to different levels of selection pressure, and how well these changes are correlated with selection of the target phenotype. The model population is composed of 857 DNAzyme clones representing 215 discrete sequence classes, which had previously been identified from two parallel selection experiments, conducted under an increasingly stringent, or permissive and constant selection time pressure. In this report, we determined the principal phenotypic properties (i.e. kobs, maximum cleavage yield and PCR efficiency) from a sample of 58 clones representing 46 different major and minor sequence classes from various rounds of each selection experiment. Interestingly, a positive correlation between the catalytic rate constant and the corresponding frequency and temporal position of a given DNAzyme was not consistently observed; however, the strength of the correlation was qualitatively higher under conditions of more stringent selection time pressure. These results suggest that the selective sampling paradigm on which in vitro selection is based, may underestimate the true functional capacity of any given random-sequence library.