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AFM study of gene silencing by DNA methylation and its interactions involving chromatin and methyl CpG binding proteins

Description

CpG methylation is an essential requirement for the normal development of mammals, but aberrant changes in the methylation can lead to tumor progression and cancer. An in-depth understanding of this phenomenon can provide insights into the mechanism of gene repression.

CpG methylation is an essential requirement for the normal development of mammals, but aberrant changes in the methylation can lead to tumor progression and cancer. An in-depth understanding of this phenomenon can provide insights into the mechanism of gene repression. We present a study comparing methylated DNA and normal DNA wrt its persistence length and contour length. Although, previous experiments and studies show no difference between the physical properties of the two, the data collected and interpreted here gives a different picture to the methylation phenomena and its effect on gene silencing. The study was extended to the artificially reconstituted chromatin and its interactions with the methyl CpG binding proteins were also probed.

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Created

Date Created
2012

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Poly(amino ether) based Polymeric and Nanoparticle Systems for Nucleic Acid Delivery and Bioimaging

Description

Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to

Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs generated using 1,4C-1,4Bis, a cationic polymer from our laboratory demonstrated significantly higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Additionally, sub-toxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ('shRNA plasmid') against firefly luciferase gene in order to knock down expression of the protein constitutively expressed in prostate cancer cells. The roles of poly(amino ether) chemistry and zeta-potential in determining transgene expression efficacies of PAE-GNR assemblies were investigated. The theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. The PAE class of polymers was also investigated for the one pot synthesis of both gold and silver nanoparticles using a small library poly(amino ethers) derived from linear-like polyamines. Efficient nanoparticle synthesis dependent on concentration of polymers as well as polymer chemical composition is demonstrated. Additionally, the application of poly(amino ether)-gold nanoparticles for transgene delivery is demonstrated in 22Rv1 and MB49 cancer cell lines. Base polymer, 1,4C-1,4Bis and 1,4C-1,4Bis templated and modified gold nanoparticles were compared for transgene delivery efficacies. Differences in morphology and physiochemical properties were investigated as they relate to differences in transgene delivery efficacy. There were found to be minimal differences suggestion that 1,4C-1,4Bis efficacy is not lost following use for nanoparticle modification. These results indicate that poly(amino ether)-gold nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging.

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Created

Date Created
2014