title: Tandem DNAzymes for mRNA cleavage: choice of enzyme, metal ions and the antisense effect.
abstract: The concept of DNAzyme-based gene silencing via mRNA cleavage was proposed over twenty years ago. A number of studies regarding intracellular gene silencing have been reported as well. However, questions have been raised regarding the lack of enzyme activity in physiological buffer conditions and it is being doubted that in the previously reported studies, gene silencing might be simply due to an antisense effect. In this work, two classical DNAzymes for RNA cleavage are studied using both chimeric substrates and extracted mRNA. We concluded that the activity of the 8-17 DNAzyme is much higher than that of 10-23 in the same conditions. To illustrate and compare the effect of specific cleavage versus antisense effect in the best possible way, we used tandem DNAzymes. Specific mRNA cleavage occurred with Zn(2+), while with Mg(2+), even the inactive control DNAzymes showed a similar response, suggesting that the antisense effect might be the dominating phenomenon causing gene silencing. This study has thus clarified the choice of DNAzyme sequence, the effect of metal ions and a potential source of antisense effect.
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Runjhun
authors.initials: R
authors.lastname: Saran
authors.affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Electronic address: liujw@uwaterloo.ca.
authors.forename: Juewen
authors.initials: J
authors.lastname: Liu
mesh_terms: D021881:DNA, Catalytic / Q000378:metabolism*; D020868:Gene Silencing; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D021881:DNA, Catalytic; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D008274:Magnesium; D015032:Zinc
keywords: Antisense; DNAzymes; Metal ions; RNA cleavage