Functionalized locked nucleic acid for therapeutic and diagnostic purposes


Ostergaard, Michael E. Østergaard. (2010). Functionalized locked nucleic acid for therapeutic and diagnostic purposes. Theses and Dissertations Collection, University of Idaho Library Digital Collections.

Functionalized locked nucleic acid for therapeutic and diagnostic purposes
Ostergaard, Michael E. Østergaard
Nucleic acids--Therapeutic use Nucleic acids--Diagnostic use
Conventional drugs target proteins while oligonucleotide (ON) drugs can target mRNA, which is present in a cell in a lower copy number than proteins (i.e. less drug load is necessary) and ON drug design is simple due to specific Watson-Crick base pairing (i.e. rapid drug development). ON drugs are, however, currently rarely used by patients due to limited target affinity, specificity and sub-optimal pharmacokinetics. Accordingly, chemically modified nucleotides are widely used to improve ON drug efficacy. Locked Nucleic Acid (LNA) exhibits very favorable hybridization properties, enhanced enzymatic stability and is commercial available. Hence LNA is widely used by multiple research groups and several ON drugs employing LNA modifications are currently being evaluated in clinical trials.;Herein, C5-functionalized LNA uridines, a new class of LNA analogs, were characterized. C5-functionalized LNA uridines were synthesized using a straightforward synthetic strategy employing only 2-3 additional steps compared to the highly optimized industry process for synthesizing LNA. When incorporated into ONs C5-functionalized LNA uridines exhibit favorable hybridization properties; small C5-substituents result in highly thermally stabilized duplexes which even surpass conventional LNA while exhibiting excellent target specificity. Large, hydrophobic, non-fluorescent C5-substituents lead to small changes in thermostability relative to unmodified DNA while preserving pronounced target specificity. Furthermore, the larger the C5-substituent the better the enzymatic stability, i.e. the C5-substituent is acting as a steric shield with the largest C5-functionalities (stearic acid and cholesterol) bestowing nuclease immunity to the ONs. CS-functionalized LNA uridines are thus promising building blocks for use in antisense ONs (i.e. targeting of mRNA) to optimize RNA affinity, target specificity, enzymatic stability and pharmacokinetics of the drug.;880-01Initial studies of 2'- N -(pyren-1-yl)carbonyl-2'-amino-LNA also called Glowing LNA has previously shown promise as a fluorescent probe signaling duplex formation. Preliminary data showed that Glowing LNA probes exhibit favorable target affinity, specificity and brightly fluorescent duplexes. Herein, Glowing LNA probes were further investigated by: (1) probe design optimization, (2) incorporation into a quencher-free molecular bacon, and (3) detection of RNA in a living cell. Overall, Glowing LNA probes exhibit low sequence dependency and high biostability rendering them promising probes for diagnostic purposes as well as a research tool to study RNA trafficking in cells.;This report highlights the advantages of combining short rigid linkers with conformationally restricted nucleotides to position functionalities precisely in a duplex. Precise spatial control of functionalities in a duplex will lead to improved diagnostic probes. Precise positional control, however, can also lead to improved ON drugs as rigid positioning of functionalities can have a significant influence upon interactions with proteins in vivo, which will result in altered pharmacokinetics.
Thesis (Ph. D., Chemistry)--University of Idaho, July 2010.
Major Professor:
Patrick J. Hrdlicka.
Defense Date:
July 2010.
Format Original:
xxix, 136 leaves :ill. (some col.) ;29 cm.

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