mRNA, which may interfere with the efficiency of the DNAzyme. Furthermore, the bulge structure would activate cellular RNases to hydrolyze the mRNA, resulting in increased mRNA transcript degradation. We introduced different sized oligo loops between the DNAzyme core sequence and the ASON sequence (Figure 1). ASON–Bulge–DNAzyme significantly reduced the expression of eGFP To assess the efficiency of the ASON–Bulge–DNAzyme complex, the ASON–Bulge–DNAzymes were chemically synthe- sized and co-transfected with pEGFP-C1 plasmid into BHK21 cells. As shown in Figure 2 (A, B and C), the ASON–Bulge– DNAzymes exhibited a significant reduction in enhanced green fluorescent protein (eGFP) expression compared with the regular DNAzyme. To examine whether the efficiency of the ASON–Bulge– DNAzyme was site limited, we synthesized another ASON– Bulge–DNAzyme targeting a different site, the eGFP mRNA. As shown in Figure 2 (D and E), the new structure also exhibited much higher efficiency than the regular DNAzyme. These results demonstrated that the bulge-containing DNAzyme was more effective in gene silencing compared with the regular DNAzyme. The effect of size and position of the bulge on the efficiency of gene silencing To assess the inhibition effect of different bulge constructs, we tested eGFP expression levels by a GFP fluorescence assay. DNAzymes with three, six, nine and 12 oligo bulges were designed and co-transfected with pEGFP-C1 plasmid into BHK21 cells. The results showed that the six oligo bulge was the most effective (Figure 3). Furthermore, the DNAzyme was most effective when the six oligo bulge was located 12–15 bp away from the core catalytic domain (Figure 4). ASON–Bulge–DNAzyme inhibited the expression of eGFP over a long time period To quantitatively assess the inhibition effect of these constructs, we tested eGFP expression levels at different time points by a GFP fluorescence assay. The results further confirmed that the ASON– Bulge structure could not only enhance the efficiency of the DNAzyme, but could also sustain the inhibitory effect for longer compared with the regular DNAzyme (Figure 5). ASON–Bulge–DNAzyme decreased eGFP expression via the mRNA degradation pathway To test whether the ASON–Bulge–DNAzyme decreased eGFP expression by virtue of the mRNA degradation pathway, a semi- quantitative RT-PCR assay was employed to test the mRNA level ASON-DNAzyme Complex of eGFP. The results confirmed that the ASON–Bulge structure could enhance the efficiency of the DNAzyme by decreasing the eGFP mRNA level. Western blot analysis further confirmed the efficiency of the ASON–Bulge–DNAzyme (Figure 6). ASON–Bulge–DNAzyme significantly reduced the expression of luciferase To assess whether the ASON–Bulge–DNAzyme was gene specific, we synthesized an ASON–Bulge–DNAzyme against firefly luciferase. The results showed that this structure was more effective than the regular DNAzyme (Figure 7). ASON–DNAzyme was effective against, and specific to, endogenous genes To test whether the ASON–Bulge–DNAzyme was effective against endogenous genes, we selected integrins as target molecules. Integrins are a large family of heterodimeric trans- membrane glycoproteins that attach cells to extracellular matrix proteins of the basement membrane or to ligands on other cells. There are many types of integrins on the cell surface relating to different functions. We designed an ASON–Bulge–DNAzyme against the endogenous integrin b1 (targeted sequence b1: 59- GATTCTCCAGAAGGTGGTTTCGATGCCATCATGCAAG- TTGCAGTTTGTGGATCACTGATT-39, b3: 59-GATGCCC- CAGAGGGTGGCTTTGATGCCATCATGCAGGCTACAG- TCTGTGATGAAAAGATT-39). The chemically synthesized DNAzyme was used to transfect HepG2 cells. As shown in Figure 8, the mRNA level of integrin b1 was significantly decreased, while the mRNA level of integrin b3 was unchanged compared with the regular DNAzyme. These results showed that this structure not only enhanced the efficiency of the DNAzyme, but also increased its specificity by minimizing the side effects, i.e., digestion of non-target mRNA. Discussion Although RNA interference is the most widely used tools for gene silencing, The instablity, short half-life, inducing undesired IFN responds and other drawbacks per se limits its application in biology studies, clinical trials, and antivirus treatments [26,27,28,29]. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and inherently more stable [30,31]. DNAzymes can be designed to cleave virtually any RNA target by selecting appropriate flanking sequences [32,33]. The recognition domains provide specificity and binding energy, however, if the binding strength is too strong or too weak between the DNAzyme and the substrate this can be antagonistic to catalysis [34,35,36]. Figure 1. Schematic of the structure of ASON-Bulge-DNAzyme. A. Regular DNAzyme. B. ASON-Bulge-DNAzyme. A 15 base AS-ON was inserted at the 59 end of ‘‘10-23’’deoxyribozyme to target the mRNA substrate and an unmatched loop between the DNAzyme sequence and ASON sequence was designed. doi:10.1371/journal.pone.0018629.g001 PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e18629