Inhibition of Ampicillin-Resistant Bacteria by Novel Mono-DNAzymes and Di-DNAzyme Targeted to β-Lactamase mRNA

Abstract

In view of the weakness of antibiotics and the properties of antisense drugs, we applied DNAzymes to the field of drug resistance in bacteria. Two 10-23 mono-DNAzymes (Dz1, Dz2) and a di-DNAzyme (Dz1-2) targeted to beta-lactamase mRNA were designed to determine to what degree the growth of ampicillin-resistant bacteria (TEM-1, TEM-3) was inhibited. All three DNAzymes can play a role both in vitro and in vivo. In vitro, they exhibited high catalytic efficiency (kcat/KM) of 63.5, 91.1, and 30.8 pM(-1) min(-1), respectively, under multiple-turnover conditions. In vivo, after 9 hours' incubation, the degree of inhibition of Dz1, Dz2, and Dz1-2 for TEM-1 bacteria was 27.2%, 39.6%, and 57.7%, respectively, and that for TEM-3 bacteria was 39.1%, 44%, and 62.6%, respectively. Dz1-2 showed the greatest inhibiting effect, demonstrating in vivo activity may be increased by constructing multiple-target DNAzymes. The results indicated a potential possibility for DNAzymes to act as a new type of antibacterial or a tool of gene functional analysis for prokaryocytes.

DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes

“…However, a majority of work where downregulation of gene expression was studied using inactive mutant Dzs as controls have shown that biological activity of Dzs could be unequivocally ascribed to their intrinsic ability to catalyze RNA cleavage in cells (see, e.g., [12][13][14][15][16][17][18][19][20][21]). Our understanding of the intracellular mechanism(s) of action of an oligonucleotide is crucial for successful nucleic acid drug design, in particular from the point of view of possible off-target effects.…”

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

“…Intracellular introduction of the deoxyribozyme led to a decrease in the levels of the targeted mRNA as well as the number of colony-forming units (CFUs) in a dose-dependent manner, while also increasing the antibiotic sensitivity of the treated bacteria. Studies with ampicillin-resistant Escherichia coli demonstrated the increased effectiveness of using a di-DNAzyme, which is a single DNA oligonucleotide containing two 10-23 enzyme regions with binding arms directed to two separate target sites on a single transcript [158]. Whereas single deoxyribozymes showed high catalytic activity in vitro, the di-DNAzyme approach was even more effective in vivo and led to decreased bacterial growth in the presence of ampicillin.…”

“…Target RNA for in vivo deoxyribozyme-catalyzed cleavage References Egr1 zinc finger transcription factor [139 -142] Epstein-Barr virus latent membrane protein [143] Hepatitis B virus X protein [144] Hepatitis B virus HBs and HBe antigens [145] Hepatitis C virus core protein [146] HIV-1 Gag [147 -149] HIV-1 Tat protein [150,151] HIV-1 5'-untranslated region [152] Human telomerase reverse transcriptase [153] Influenza virus A [154] Isocitrate lyase from M. tuberculosis [155] c-Jun leucine zipper transcription factor [156,157] b-Lactamase [158,159] 12-Lipoxygenase [160] c-Myc proto-oncogene [161 -163] Ornithine decarboxylase [164] Pencillin-binding protein [165] PML/RARa fusion gene of acute promyelocytic leukemia [166] Respiratory syncytial virus (RSV) nucleocapsid protein [167] SARS associated coronavirus 5'-untranslated region [168] Survivin [169] TGF-b1 [170] Twist helix-loop-helix transcription factor [171] Vascular endothelial growth factor receptor 2 (VEGFR) [172] This tabulation is representative, not comprehensive. Additional examples and reviews are cited in ref.…”

Bioinformatics Designing of 10-23 Deoxyribozyme against Coding Region of Beta-galactosidase Gene

“…TEM-1 and TEM-3 (5) and Methicillin-resistant Staphylococcus aureus (MRSA) (6)(7)(8). Effective inhibition of ampicillin-resistant bacteria TEM1 and TEM3 has been demonstrated by novel mono-Dzs Dz1, Dz2, and bis-Dz Dz1-2 targeting β-lactamase mRNA (8). The same strategy has been used by Hou and Unwalla in recent years (9,10).…”

“…Dzs were investigated against ampicillin-resistant E. coli strains with extended spectrum β-lactamase i.e. TEM-1 and TEM-3 (5) and Methicillin-resistant Staphylococcus aureus (MRSA) (6)(7)(8). Effective inhibition of ampicillin-resistant bacteria TEM1 and TEM3 has been demonstrated by novel mono-Dzs Dz1, Dz2, and bis-Dz Dz1-2 targeting β-lactamase mRNA (8).…”