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Towards intracellular aptamers: delivery of anti-SCV helicase aptamers and development of aptamers againstSATB1Kimura, Mari, 木村摩利 January 2012 (has links)
Aptamers are small RNAs or DNAs that specifically bind to targets through complementary three-dimensional structure with high affinity. Aptamers are screened by an in vitro process called SELEX (systematic evolution of ligands by exponential enrichment) against a variety of targets, including small organic and inorganic molecules, cofactors, peptides, proteins and even whole cells, and aptamers hold great potential as diagnostic tools or as therapeutics. Aptamers are alternatives to antibodies with a variety of potential advantages. However, development of aptamers against intracellular targets is limited by delivery methods. To develop an intracellularly acting aptamer, we aimed to 1) establish an aptamer intracellular delivery system; 2) clone, express and purify the intracellular target SATB1 for aptamer selection; and 3) select an intracellularly acting aptamer against SATB1 by SELEX.
An efficient delivery for the anti-SCV helicase aptamer was achieved using the pDNA transfection reagent Lipofectamine2000, whereby the aptamer was delivered exclusively to the nucleus. We also tested and improved methods to study aptamer-liposome complex formation. Expression of the SCV helicase in the cytoplasm could not alter the aptamer location within cell, suggesting that aptamer modification such as attachment of a redirecting signal or conjugation to a polymer would be required for cytoplasmic targeting. In this thesis we switched to a nuclear target, SATB1, to develop a nuclear intracellularly acting aptamer.
SATB1 is a gene regulator found in the nucleus. Upon activation, SATB1 binds to the nuclear matrix and targets the chromosome via the MAR binding domain to regulate histone modification and nucleosome positioning over a long distance. A recent report demonstrated SATB1’s role in breast cancer metastasis, therefore development of aptamers against this protein has great diagnostic and therapeutic potential. We successfully cloned full length SATB1. The full length protein could not be expressed, however the MAR binding domain was successfully expressed with 6xHis tag and purified using a His trap column. ITC analysis with BUR sequence showed proper folding and selectivity of the MAR binding domain. DNA aptamers were selected by SELEX against the MAR binding domain of SATB1. Selection was successful and a highly conserved family of aptamers was observed. Sequence analysis and alignment revealed the presence of many conserved motifs that involve many A and T in a similar manner to the BUR sequence and dsDNA previously found to have high affinity towards the MAR binding domain.
Altogether, we have taken a step closer towards the development of an intracellularly acting SATB1 aptamer. Future efforts involving affinity determination, application of the established delivery method and in vitro study of inhibitory mechanism will be further steps towards intracellular aptamers for cancer diagnosis or therapeutics. / published_or_final_version / Biochemistry / Master / Master of Philosophy
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Synthesis, selection, and characterization of oligonucleotide hybrids with increased target affinity and selectivity /Bleczinski, Colleen F. January 2000 (has links)
Thesis (Ph.D.)--Tufts University, 2000. / Adviser: Clemens Richert. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 140-151). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Pharmacokinetics, hepatic extraction, and renal disposition of phosphorothioate oligodeoxynucleotidesCho, Min-hee. January 2002 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.
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Advances in solid phase synthesis of neutral oligonucleotide analoguesWang, Haiyan, 1963- 26 November 1991 (has links)
A novel acyl protecting group for cytosine and adenine has been prepared
from 4-( chloromethyl) benzoic acid. Reaction of the acid with morpholine produces
4-( 4-morpholinyl )methyl benzoic acid which is converted to it's acid chloride
with thionyl chloride. This may be used to acylate cytidine and adenosine under
standard conditions. This ionizable protecting group has the ability to solublize
protected oligomers, which allows their purification with ion-exchange chromatography
on S-Sepharose. Solid phase synthesis has been performed using this protecting
group on morpholine nucleosides. Morpholine nucleoside carbamates were
synthesized in high yields. The results revealed that high purities of the hexamers
were obtained. These hexamers, which have the protecting groups intact, provide
the potential for further segment condensation to make large size oligonucleotide
analogues.
The success of the solid phase synthesis was dependent upon use of a selectively
cleavable anchor which allowed the finished oligomer to be released from the resin
with protecting groups intact. Attempts to modify the anchor to make it more efficient
were unsuccessful. It was found that DBU degrades derivatized polystyrene
resin and should not be used at early stages in solid phase synthesis. / Graduation date: 1992
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Mutagenic properties of psoralen-modified triple helix forming oligonucleotidesSándor, Zoltán. January 1998 (has links)
Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.
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Mutagenic properties of psoralen-modified triple helix forming oligonucleotidesSándor, Zoltán. January 1998 (has links)
Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.
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X-ray diffraction studies of crystalline DNAHubbard, Steven Robert January 1994 (has links)
No description available.
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Inhibition of oncogene expression by the formation of Triplex DNARichards, Sally January 2001 (has links)
No description available.
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Design of B-form specific DNA binding oligonucleotide analogues : investigations of novel recognition moieties for triple helix formationHogeland, Jane S. 01 June 1993 (has links)
Graduation date: 1994
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Solid-phase synthesis and biophysical testing of uncharged acyclic oligonucleotide analoguesNelson, Jeffrey Stephen, 1962- 23 February 1994 (has links)
Graduation date: 1994
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