Enforcing DNA duplexes with molecular caps

Reported here are the results of a study on the stabilizing effect of carboxylic acid residues appended to thymidine or uridine residues at the termini of oligonucleotides on DNA duplexes formed by these oligonucleotides. Both appendages at the 5′- and 3′-terminus of oligonucleotides were studied. These were introduced through peptide coupling reactions between protected aminodeoxyoligonucleotides and carboxylic acids. Increases in duplex stability were demonstrated in UV-melting experiments, where melting temperatures of unmodified control duplexes and duplexes with modified terminal nucleotides were determined. In order to screen combinatorial libraries of modified oligonucleotides rapidly, mixtures of the test strands and one equivalent target strand were exposed to single strand-specific nucleases. Snake venom phosphodiesterase (E.C. 3.1.4.1) was employed for 5′ -modified oligonucleotides, and calf spleen phosphodiesterase (E.C. 3.1.16.1) for 2′/3′-modified strands. MALDI-TOF mass spectrometry was used to monitor these nuclease selection experiments, in which rapid disappearance of full length oligomers indicated low affinity for the target strand.; The synthesis of the 5′-modified oligonucleotides largely followed a route recently established in these laboratories. This route involves the conversion of thymidine into 5′-amino-5 ′-deoxythymidine via tetrachlorophthalimide or azide intermediates, 5′-protection, 3′-phosphitylation, and solid phase synthesis. For the synthesis of oligonucleotides with appendages at the 3′-terminus, a new synthetic route was developed. On this route, 2′-amino-2′-deoxyuridine, prepared in three steps from uridine, was doubly protected, using an Fmoc group for the amino function, and an isopropylidine group for the 2 ′- and 3′-functionalities, and phosphitylated at its 5′-hydroxyl group. Solid phase synthesis with this and other ‘reversed’ phosphoramidites then furnished a protected oligomer, whose terminal isopropylidene group was made labile through removal of the Fmoc-group. Unlike the route developed earlier, which employs conventional phosphoramidites and thus a placement of the modified residue close to the surface of the solid support, the new method gave high yields of acylated products and allowed the generation of combinatorial libraries via mixed coupling reactions. (Abstract shortened by UMI.)...