Silencing Transcription: Promoter-Targeted Oligonucleotides Bind Chromosomal DNA Inside Cells

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Silencing Transcription: Promoter-Targeted Oligonucleotides Bind Chromosomal DNA Inside Cells

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Title: Silencing Transcription: Promoter-Targeted Oligonucleotides Bind Chromosomal DNA Inside Cells
Author: Beane, Randall L.
Abstract: Aberrant gene expression can lead to multiple disease-states that can be difficult or impossible to treat using traditional small-molecule medications. An alternative approach to treating such diseases is oligonucleotide-based therapeutics, which are theoretically capable of treating or curing genetic diseases, infections, and abnormalities. Oligonucleotide-based molecules targeted to DNA are referred to as antigene agents. These molecules can silence or activate gene transcription of alleles and have many potential medical applications. However, the growth of antigene technologies has been slow despite broad therapeutic potential and unique molecular applications. Through the development of chemical modifications, oligonucleotide-based molecules are actively being improved and refined. Chemical modifications can alter the cellular uptake, toxicity, biodistribution, and plasma retention of oligonucleotides. My research goal was to further the field of synthetic antigene oligonucleotides. To do this, I targeted endogenous genes in human cancer cell lines with chemically-modified oligonucleotides, including MOEs, PNAs, ENAs, and LNAs. I established that LNAs were robust antigene agents capable of inhibiting transcription under multiple conditions. Specifically, I established that mixed-base antigene agents physically associate with the hPR-B promoter and decrease the occupancy of RNA polymerase II on the hAR and hPR genes inside human cells. Furthermore, my research indicates that antigene LNAs function in an orientation-dependent manner and that functional LNAs must target the template strand of DNA to have appreciable potency. This body of work comprised the first extensive study of a mixed-base antigene oligonucleotide in multiple human cell lines and provides the first evidence that mixed-base antigene agents can physically associate with chromosomal DNA and inhibit transcription of endogenous mammalian genes inside human cells. Collectively, my data suggest that antigene LNAs are a potent and general strategy for silencing gene expression, and that antigene LNAs also have potential therapeutic applications and possible utility in modern functional genomics.
URI: http://hdl.handle.net/2152.5/673
Date: 2009-01-08

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