ASON-DNAzyme Complex Figure 2. Transfection of ASON-Bulge-DNAzyme resulted in significantly reduced eGFP expression. BHK21 cells were co-transfected with 0.5 mg pECFP-C1 plasmid and 0.25 mg of DNAzymes A. pEGFP control; B. Regular DNAzyme-1; C. ASON-Bulge-DNAzyme-1; D. Regular DNAzyme-2; E.ASON-Bulge-DNAzyme-2. Calibration bar for A-E, 100 mm. doi:10.1371/journal.pone.0018629.g002 PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e18629 Figure 3. The effect of different bulge size on DNAzyme efficiency. Cells were cotransfected with 0.5 mg pECFP-C1 plasmid and 0.25 mg of DNAzymes. 1. pEGFP control; 2. Regular DNAzyme; 3. 3nt- Bulge-DNAzyme; 4. 6nt-Bulge-DNAzyme; 5. 9nt-Bulge-DNAzyme; 6. 12nt-Bulge-DNAzyme. The error bars denote Standard deviation (SD). doi:10.1371/journal.pone.0018629.g003 DNAzymes face the same challenges of ribozymes such as the ability to be targeted to the cell of interest, intracellular localization, the ability to hybridize with the mRNA target, and the ability to achieve a sufficient intracellular concentration while avoiding interacting with non-target mRNAs [37,38]. Within living cells, mRNA transcripts exist in secondary structures and interact with cytoplasmic proteins, therefore, some of the mRNA sequences are hidden and only partial sequences are accessible [39]. mRNAs exist in several cellular compartments, including the cytoplasm, nucleus and nucleolus. To suppress gene expression, DNAzymes must be colocalized to the same intracellular compartment as their target mRNA [40,41]. Antisense RNA inserted within a variable region of ribosomal RNA (rRNA) has been shown to enhance ribozyme efficiency due to colocalization of rRNA with mRNA [42,43]. In this study, we introduced complementary oligonucleotides (ASONs) at the 59 end of DNAzymes to enhance the specificity of targeting to the mRNA substrate. To avoid too high a binding strength between the DNAzyme and the target mRNA, we introduced a nonpairing bulge structure into the DNAzyme. The formation of a bulge structure ensured that the target mRNA was easily hydrolyzed. Our results demonstrated that the incorporation of an ASON and a mispairing bulge in the DNAzyme conferred higher efficiency and specificity in gene silencing. Materials and Methods Design and synthesis of the ASON–Bulge–DNAzyme A 15 base ASON was inserted at the 59 end of the ‘‘10-23’’ deoxyribozyme. To avoid too high a binding strength between the DNAzyme and the target mRNA which might interfere with