Inhibition Of Bacterial Translation And Growth By Peptide Nucleic Acids(PNAs) Targeted To Domain Ⅱ Of 23S RRNA

Antibiotics are widely used in medicine,agriculture and industrial fermentations. However,bacterial drug resistance has become a serious problem,and research on new antibiotics is very slow.Current problems with resistance arose in part because nearly all antibiotics used in clinic are derived from natural substances of micro origin,and microbs resident in the environment possess corresponding resistance genes.Many studies, including antisense inhibition,are going on to conquer microbes.Antisense inhibition is a basic mode to regulate gene expression in micro origins. Functional domains of mRNA and rRNA are the most common entry targets for antisense regulation.Translational inhibition by antisense oligonucleotide technology is very promising,but there are still many problems,e.g.sequence specificity,biological stability, solubility and cell uptake.Peptide nucleic acid(PNA) is a DNA mimic that bears a pseudo-peptide backbone composed of achiral and uncharged N-(2-aminoethyl) glycine units.PNA has been shown to hybridize in a sequence-specific manner and with high affinity to complementary sequences of single-stranded DNA or RNA molecules,while resistant to digestion and degradation by nucleases and proteases.Homopyrimidine PNAs bind to complementary DNA/RNA targets forming(PNA)₂-DNA/RNA triplexes of very high thermal stability,two PNA chains of which combine with DNA/RNAfollowing the base pairing rules of Watson-Crick and Hoogsteen.The potential of PNAs as specific inhibitors of translation has been studied,and it has been shown that PNAs targeted to domain V of 23S rRNA and mRNA can significantly inhibit protein synthesis in bacteria.In 23S rRNA domainâ…¡,G1138,C1045 and A1067 sites are associated with GTPase and are close relatives to the rRNA peptidyl transferase center and the EFG elongation factor.Adjacent sequences of G1138,C1045 and A1067 sites are single-stranded.Hanvey et al.have demonstrated that in cells PNAs are difficult to bind with dsDNA because of high ionic concentration,but they can form highly stable hybrid compounds with single stranded DNA.PNA is inefficiently taken up by cultured cells and is slow to move across phospholipids vesicle(liposome) membranes.Furthermore,the molecular weight of typical oligonucleotides exceeds the expected cut-off for efficient passive diffusion through the nonspecific porin channels that span the E.coli cell wall.However,attachment of PNAs to the cell-permeabilizing peptide KFFKFFKFFK can dramatically improve antisense potency.In this study,PNA(G1138),PNA(A1067) and PNA(C1045) were designed.Their effects on translation inhibition were studied in a cell-free translation system.PNAs that can effectively inhibit translation in vitro were then designed as peptide (KFFKFFKFFK)-PNAs to analyze their effects on suppressing E.coli growth in vivo.Sectionâ… The expression of soluble native EGFP in E.coli and Cell-Free translation systemObjective To construct the pET28a-EGFP plasmid which can heavily express the soluble native EGFP in E.coli BL₂₁(DE₃) and cell-free translation system.Methods Using pEGFP-N1 plasmid as template,full-length EGFP cDNA was amplified by polymerase chain reaction(PCR).The amplified EGFP cDNA was cleaved with EcoR I and Hindâ…¢, the PCR fragment was gel purified and ligated into pET28a plasmid cleaved by EcoRI and Hindâ…¢using T₄ DNA ligase,then the ligated products were transformed into E.coli DH5α. The positive clone was propagated and the recombinant plasmid was extracted for further identification and sequencing.The correct recombinant plasmid was named pET28a-EGFP. E.coli BL₂₁(DE₃) was transformed with the recombinant pET28a-EGFP,and induced with IPTG for protein expression.EGFP in E.coli BL₂₁(DE₃) was identified by Western blotting and the fluorescence was observed by the fluorescence microscope.The recombinant pET28a-EGFP was added into the cell-free translation system,and incubated two hours. EGFP in cell-free translation system was identified by Western blotting which first antibody were EGFP antibody,T7-tag antibody and His-tag antibody,respectively.The fluorescence in cell-free translation system was observed by the fluorescence microscope. Results It was confirmed by DNA sequencing that the EGFP cDNA sequence in recombinant plasmid pET28a-EGFP was in accordance with the EGFP cDNA sequence in GenBenk.The fluorescence was saw by the fluorescence microscope in E.coli BL₂₁(DE₃) and cell-free translation system.Western blotting demonstrated that the high efficient expressed protein in E.coli BL₂₁(DE₃) and cell-free translation system was native EGFP which molecular weight is about 27kd.Conclusions The recombinant pET28a-EGFP plasmid which can heavily express the soluble native EGFP in E.coli BL₂₁(DE₃) and cell-free translation system were constructed successfully,which provides a reporter molecule to screening the bacteriostatic agents in vitro and a basis to further establish a standard substance of fluorescence intensity.Meanwhile,a new conception was provided to design primer.Sectionâ…¡Inhibition of bacterial translation by peptide nucleic acids(PNAs) targeted domainâ…¡of 23SrRNA in free-cell translation systemObjective In this section,we have attempted to study the inhibitory effects of peptide nucleic acids targeted 23SrRNA domainâ…¡on bacterial translation in cell-flee translation system.Methods Firstly,we chose EGFP as the reporter molecule,after recombinant plasmid pET28a-EGFP and PNAs targeted to 23 SrRNA domainâ…¡were incubated together in cell-free translation systems,the decline of fluorescence intensity of EGFP was observed and determined by the fluorescence microscopy and fluorospectrophotometer.Secondly,to qualitatively demonstrate that the decline of fluorescence intensity was due to the reduction of EGFP expression,we adopt more sensitive autoradiography to analyze the expression of EGFP.In addition,we measured the relative incorporation of[³⁵S]methionine by TCA protein precipitation assay to quantitatively calculate the inhibitory concentrations(IC₅₀) of PNAs.Finally,we utilized the turbidity assay to study the bacteriostasis of PNA(G1138) in MH broth.Results PNA(G1138) targeted 23SrRNA domainâ…¡could inhibited bacterial protein translation in cell-free translation system in a dose- and sequence-dependent manner,the inhibition effect of PNA(G1138) in vitro is comparable to that of tetracycline, the inhibitory concentrations(IC₅₀) of PNA(G1138) and tetracycline are 0.15μmol/L and 0.12μmol/L,respectively.The bacteriostatic effect of PNA(G1138) in MH broth was not conspicuous.The minimum inhibiting concentration(MIC) of PNA(G1138) is＞50μmol/L. Conclusions The G1138 site of 23S rRNA is a ideal sequence target for designing novel PNA-based antibiotics.PNA(G1138) can not been inefficiently taken up by cultured E.coli DH5α.Sectionâ…¢Inhibition of bacterial growth by peptide-PNAs targeted to domainâ…¡of 23S rRNAObject To further study the bacterial inhibitory growth of peptide-PNA(G1138). Methods The bactericidal effect of antibacterial peptide-PNA(G1138) conjugates on E.coli DH5αwere observed by turbidity analysis and were further examined by a cell viability study.Results peptide-PNA(G1138) targeted to domainâ…¡of 23S rRNA can inhibit bacterial growth in a dose- and sequence-dependent manner,but the minimum inhibiting concentration(MIC) of peptide-PNA(G1138) against E.coli is significantly higher than that of tetracycline,the bactericidal effect is less effective than that of tetracycline.Their minimum inhibiting concentrations(MICs) are 10μmol/L and 4μmol/L,respectively. Conclusions peptide-PNA(G1138) at 10μmol/L can inhibit E.coli DH5αgrowth,but the inhibitory effect of peptide-PNA(G1138) is much higher than that of tetracycline and other PNAs targeted to domain V of 23S rRNA.To screen more effective cell-permeabilizing peptide and to improve the bactericidal effect of PNA(G1138) are the aims of further research.