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Design and cloning of hammerhead ribozymes targeted to NL43 HIV-1 Vif MRNA.

HIV, the etiological agent of the Aquired Immunodeficiency Syndrome (AIDS) poses a worldwide threat to public health. The ability of HIV to adapt to novel antiviral drugs makes gene therapy and the use of ribozymes a promising avenue of research in the battle against AIDS. Hammerhead ribozymes are RNA molecules that act as enzymes, catalyzing the substrate-specific cleavage of substrate RNA. Ribozyme targeting HIV RNAs could potentially inhibit viral replication, reducing HIV expression. Vif (Virion Infectivity Factor), one of fifteen distinct proteins encoded by the HIV genome, enhances viral infectivity by blocking a host antiviral defense pathway that relies on the host protein APOBEC3G. Vif's ability to inhibit this cellular retroviral defense mechanism makes it a potentially advantageous target for ribozyme-mediated down-regulation. A catalytic and a non-catalytic(A) hammerhead ribozyme targeted to a GUU at nucleotide 5154 of the Vif sequence were designed based on the model of Haseloff and Gerlach (Nature, 1988). These ribozymes differed by a single nucleotide in the catalytic core. These ribozymes were synthesized and used as templates in PCR reactions to generate double-stranded DNA. Each was cloned into pPCR-Script by blunt-end ligation and transformed into XL-10 Gold Ultracompetent cells. Several colonies were analyzed for the presence of ribozyme-positive plasmid DNA by PCR using an M13 primer mix. Ribozyme orientation was determined by PCR using M13 and ribozyme-specific primers. Two clones were subsequently sequenced to verify ribozyme presence. These plasmids, pVif5154 Rz and pVif5154A Rz will be used in in vitro cleavage reactions to measure ribozyme kinetics. Pending the outcome of these tests, future studies may also include tissue culture testing of these ribozymes * Supported by NIH Grant 1R15 GM66678-01
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Author:Anderson, Katherine L.; Jackson, William H.
Publication:Bulletin of the South Carolina Academy of Science
Date:Jan 1, 2005
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