Synthesis, structure, and activity of hybridization probes and immunostimulatory DNA

This doctoral thesis reports on the results from two different projects involving synthetic oligonucleotides. The oligonucleotides were generated to study their properties as hybridization probes or as immunostimulatory agents. The first part of this thesis focuses on oligonucleotides with molecular caps as fidelityenhancing elements for hybridization probes. The molecular basis of the duplex-stabilizing effect of an anthraquinonyl (AQ) residue at the 2'-position of a 3'-terminal 2'-amino-2'- deoxyuridine was determined through a high resolution structure, obtained through twodimensional NMR and restrained molecular dynamics. The three-dimensional structure of (ACGCGU-AQ)2 revealed that the anthraquinone moiety does not bridge the duplex with an intact terminal A: U base pair, but rather disrupts this base pair, capping the penultimate G: C base. Based on this structural information, a „composite cap“ encompassing both the uridine residue and the anthraquinonyl group was designed, in which the anthraquinone can bridge terminal base pairs and the uridine residue helps to seal them off from the minor groove. The composite cap was then shown to be the most strongly duplex-stabilizing cap for the 3'- terminus of DNA hybridization probes to date, inducing enhanced affinity and base pairingselectivity in duplexes with complementary DNA and RNA strands. The second part of this thesis focuses on oligonucleotides that stimulate the innate immune system. A range of oligonucleotides containing a phosphodiester backbone and the core sequence 5'-GACGTT-3' were prepared that have the propensity of forming higher ordered structures. The structures were believed to enhance the immunostimulatory effect by affecting cellular uptake and recognition by the receptor. Also, the structure of the wellvii known immunostimulatory DNA eicosamer 5'-TCCATGACGTTCCTGATGCT-3' and shortened or extended versions thereof, ranging in length from decamer to pentaeicosamer were investigated by 1H NMR spectroscopy. The spectra suggested that the immunostimulatory oligonucleotides form intermolecular duplexes. Structures are formed beyond the core hexamer GACGTT that appear to involve the 5'-terminus.