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
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/161584 |
| Date | January 2012 |
| Creators | Kimura, Mari, 木村摩利 |
| Contributors | Tanner, JA, Huang, J |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B48079893 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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