Efficient Silencing of Gene Expression by an ASON– Bulge–DNAzyme Complex Jianzhong Yi*, Chengqian Liu Institute of Animal Husbandry Veterinary Sciences, Shanghai Academy of Agricultural Sciences, Shanghai, China Abstract Background: DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The ‘‘10-23 DNA enzyme’’ is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs. Methodology: In this study, we inserted a nonpairing bulge at the 59 end of the ‘‘10–23 DNA enzyme’’ to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 59 end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme. Conclusions: Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12– 15 bp away from the core catalytic domain. Citation: Yi J, Liu C (2011) Efficient Silencing of Gene Expression by an ASON–Bulge–DNAzyme Complex. PLoS ONE 6(4): e18629. doi:10.1371/ journal.pone.0018629 Editor: Joseph Najbauer, City of Hope National Medical Center and Beckman Research Institute, United States of America Received December 22, 2010; Accepted March 14, 2011; Published April 7, 2011 Copyright: 2011 Yi, Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was sponsored by the Pujiang Program from Science and Technology Commission of Shanghai Municipality (Project no. 07pj14074). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yijianzhong@yahoo.com Introduction At least four different approaches have been used for gene silencing to date, these include: gene knockout by homologous recombination [1]; antisense inhibition by the use of synthetic oligonucleotides that hybridize with DNA or RNA [2,3,4]; cleavage of target mRNA by ribozymes or DNAzymes [5]; and RNA interference by inducing the endogenous siRNA process pathway [6,7]. Studies by Paterson et al. (1977) demonstrated that gene expression could be modified through the use of exogenous nucleic acids that were complementary to target RNAs. Zamecnik and Stephenson (1978) inhibited the replication of Rous sarcoma virus by the use of a short antisense DNA oligonucleotide (ASON) that was reverse complementary to a particular transcript of the virus [8]. ASONs or ASON analogs are often employed for in vivo antisense applications due to their stability and nuclease resistance [9,10,11]. Generally, these molecules block gene expression by hybridizing to the target mRNA, resulting in subsequent double- helix formation which inhibits transcript processing or translation. Furthermore, cellular RNases are able to bind to the DNA–RNA duplex and hydrolyze the mRNA, resulting in increased mRNA transcript degradation [12,13,14]. Breaker and Joyce (1994) made the assumption that because RNA and DNA are similar chemical compounds, DNA molecules with enzymatic activity could also be developed as ribozymes. This proposition led to the development of a DNA enzyme identified from a library of .1,000 different DNA molecules by successive rounds of in vitro selective amplification. The selected molecule was designated the ‘‘10–23 DNA enzyme,’’ and was composed of a catalytic domain of 15 deoxynucleotides flanked by two substrate-recognition domains of ,8 nucleotides on each side [15,16,17]. The recognition sequences provided the specificity for binding to target RNA substrates and supplied the binding energy required to hold the RNA substrate with the DNAzyme [18,19]. Although DNAzymes are less costly and more stable than ribozymes, they still encounter problems such as stability, efficiency and specificity to target mRNAs [20,21]. Their complexes with RNA are less stable than the corresponding RNA:RNA duplexes, hence, mismatches might be generated during their interactions with non-target mRNAs, such as the mRNAs from other members of a gene family, resulting in non-specific cleavage [22,23,24,25]. In this study, we introduced new bulge structure at the 59 end of the ‘‘10–23’’ deoxyribozyme to enhance the efficiency and specificity of the DNAzyme. Results Design and synthesis of ASON–Bulge–DNAzymes In this study, we designed different sizes of bulges at different positions at the 59 end of the regular DNAzyme. A non-matching bulge will avoid strong binding between the DNAzyme and target PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18629