Figure 4. The effect of different bulge positions on DNAzyme efficiency. Cells were co-transfected with 0.5 mg pECFP-C1 plasmid and 0.25 mg of DNAzymes. 1. pEGFP control; 2. Regular DNAzyme; 3. 6nt; 4. 9nt; 5. 12nte; 6.15 nt. The error bars denote Standard deviation (SD). doi:10.1371/journal.pone.0018629.g004 DNAzyme efficiency, we introduced a nonpairing loop at the 59 end of the DNAzyme. The oligonucleotides used for this were as follows: Regular DNAzyme-1 against eGFP: Figure 5. Quantitative assessment of the silencing efficiency of ASON-Bulge-DNAzyme by GFP fluorescence assay. BHK21 cells were harvested and lysed in 200 ml lysis buffer (0.1 M Tris-HCl, 0.1% Triton X-100, 2 mM EDTA, pH 7.8). Thefluorescence of eGFP was measured using a luminometer. The error bars denote Standard deviation (SD). doi:10.1371/journal.pone.0018629.g005 ASON-DNAzyme Complex 59-CTCAGGTAGGGCTAGCTACAACGAGGTTGTCGG-39 ASON-Bulge-DNAzyme-1 against eGFP: 59-TGCTCAGGGCGGACTGAAGTACCTCAGGTAGGG- CTAGCTACAACGAGGTTGTCGG-39 Size of the bulge in the annealing arm complex of the DNAzyme Bulges of differing sizes were incorporated into the complex using the following oligonucleotides: The 3 nt Bulge–DNAzyme: 59-TGCTCAGGGCGGACTGTACCTCAGGTAGGGCTA- GCTACAACGAGGTTGTCGG-39 The 6 nt Bulge–DNAzyme: 59-TGCTCAGGGCGGACTGAAGTACCTCAGGTAGGG- CTAGCTACAACGAGGTTGTCGG-39 The 9 nt Bulge–DNAzyme: 59-TGCTCAGGGCGGACTGAAGCGATACCTCAGGTAG- GGCTAGCTACAACGAGGTTGTCGG-39 The 12 nt Bulge–DNAzyme: 59-TGCTCAGGGCGGACTGAAGTGACGTTAACCTCAG- GTAGGGCTAGCTACAACGAGGTTGTCGG-39 Position of the bulge in the annealing arm of the DNAzyme The position of the bulge in the complex was altered using the following oligonucleotides: 1. Bulge–6 nt–DNAzyme: 59-TGCTCAGGGCGGACTGAAGTACCTCAAGGGCTAG- CTACAACGAGGTTGTCGG-39 2. Bulge–9 nt–DNAzyme: 59-TGCTCAGGGCGGACTGAAGTACCTCAGGTAGGG- CTAGCTACAACGAGGTTGTCGG-39 3. Bulge–12 nt–DNAzyme: 59-CTTGCTCAGGGCGGAAGTACGGTGCTCAGGTAGG- GCTAGCTACAACGAGGTTGTCGG-39 4. Bulge–15 nt–DNAzyme: 59-TCTTTGCTCAGGGATCTACCTGGGTGCTCAGGTA- GGGCTAGCTACAACGAGGTTGTCGG-39 DNAzymes against Luciferase were constructed using the following oligonucleotides: Regular DNAzyme against Luciferase: 59-AGCATCGAGAGGCTAGCTACAACGACCGTTGT-39 ASON–Bulge–DNAzyme against Luciferase: 59-GGGCAATTTACCACGAGCAATAGCATCGAGAGGC- TAGCTACAACGACCGTTGT-39. DNAzymes against integrin b1 were constructed using the following oligonucleotides: Regular DNAzyme against integrin b1: 59-TTGCATGAGGCTAGCTACAACGAGGCATCG-39 ASON–Bulge–DNAzyme against integrin b1: 59-GTGATCCACAAACTGCAAGTACAACTTGCATGAG- GCTAGCTACAACGAGGCATCG-39 Semi-quantitative RT-PCR analysis of eGFP Two pairs of specific primers were designed according to the sequence of the eGFP gene and the b-actin gene. The primers were synthesized by Shanghai Generay Biotech Co., Ltd (China). eGFP-F1: 59-CTGGACGGCGACGTAAACGG-39 eGFP-R1: 59-CACGAACTCCAGCAGGACCAT-39 b-actin-F1: 59-GGCTCCGGCATGTGCAAGGC-39 b-actin-R1: 59-GAAGGTAGTTTCGTGGATGCC-39 PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18629