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    Studies on human ribonuclease H1 and its action on 2'-fluoroarabinose oligonucleotide hybrid substrates

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    Genre
    Thesis/Dissertation
    Date
    2012
    Author
    Alla, Nageswara Rao
    Advisor
    Nicholson, Allen W.
    Committee member
    Tuszynski, George P.
    Andrade, Rodrigo B.
    Wunder, Stephanie L.
    Department
    Chemistry
    Subject
    Chemistry
    Biochemistry
    2'-fana
    Antisense
    C-myb
    Rnase H1
    Permanent link to this record
    http://hdl.handle.net/20.500.12613/666
    
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    DOI
    http://dx.doi.org/10.34944/dspace/648
    Abstract
    Ribonuclease H1 is a conserved enzyme that is localized in the nuclear and mitochondrial compartments of eukaryotic cells, and functions in DNA replication, repair, recombination and transcription. (Arunachandran et al., 2000; Cerritelli et al., 2003) Oligonucleotide binding to a complementary RNA sequence can provide a substrate for RNase H1, and provides the mechanistic basis for antisense oligonucleotide (AON)-directed gene silencing in cells (Opalinska et al., 2002). Effective evaluation of the therapeutic efficacy of next-generation AONs with novel structures requires an in vitro system involving, purified, highly active RNase H1 of human cells, and a full understanding of the catalytic mechanism of the enzyme. The goal of project 1 described in chapter 3, was to determine the involvement of a conserved Histidine (H264) in the catalytic mechanism of human RNase H1. Based on this analysis I was able to conclude that H264 has a dual role in phosphodiester hydrolysis and in product release. The goal of project 2 (Chapter 4) was to examine the reactivities towards human RNase H1 of model hybrid substrates containing specific types of 2'-FANA substitutions (abbreviated as `F', with 2'-deoxyribose abbreviated as `D'), either at the "wings" of the molecule ("7-gapmer"; each wing=7 nt: FFFFFFF-DDDDDDD-FFFFFFF), or with 3 nt alternations ("3-altimer": FFF-DDD-FFF-DDD-FFF-DDD-FFF). The results of this study strongly support the continued examination of the potential therapeutic utility of the 2'-FANA modification in AONs. The highly efficient and selective inhibition of protein expression is a primary basis of action of most antisense therapeutic strategies. These data suggest that the 2'-FANA modification supports sustained silencing after a single administration, either by mRNA cleavage or by a translational block, and at substantially lower concentrations compared to the unmodified AON. The results of this project underscore the proposal that 2'-FANA-modified AONs will be important additions to the repertoire of rational antisense strategies for the effective treatment of disease.
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