Interfacial oligonucleotide chemistry studied by an on-line biosensor, radiochemical labelling and nucleic acid microarrays

This thesis presents the application of the thickness shear-mode (TSM) acoustic wave sensor to the study of interfacial oligonucleotide interactions. Radiochemical analysis of the TSM devices was performed and contrasted with confocal microscope analysis of the system immobilized onto glass slides.; Initial studies involved the investigation of HIV-1 TAR RNA and Tat peptide interaction. A 31-base sequence of the TAR RNA was immobilized to the TSM surface and challenged with a 12-amino acid peptide derived from the Tat protein such that the binding sites of both were incorporated. This was studied using both 32P-labelled TAR RNA and 125I-labelled peptide and compared with previously obtained data from the TSM sensor. The results showed that the TSM sensor is sensitive to actual TAR RNA/Tat peptide binding interactions occurring at the interface; nonspecific binding of peptide was verified by radiochemical experiments, however, this was not observed with the TSM sensor.; The hybridization of a biotinylated 25-mer oligonucleotide probe with complementary, non-complementary and single-base mismatch 25-mer oligonucleotide targets at the liquid-solid (neutravidin-modified) interface of a thickness-shear mode acoustic wave device was studied. Under ambient temperature conditions, different signals were obtained for the complementary and non-complementary cases. For non-complementary interactions, the system exhibits behaviour characteristics of the production of intermediate duplexes, which are decomposed by the re-introduction of buffer solution. The use of higher temperatures has the potential to permit the distinct on of binding events involving a set of single-base mismatch 25-mers. The different responses observed were dependent on both the nature and the location of the instigated mismatch.; Regeneration of the probe-modified surface was achieved using λ-exonuclease, an enzyme that digests a single strand of double-stranded DNA starting from the 5′-end, cleaving single nucleotides as it progresses. Thus, with the 5′ end of the probe strand inaccessible through he biotin-neutravidin linkage, the target strand was effectively removed by λ-exonuclease digestion. This was confirmed by a series of radiochemical experiments involving oligonucleotides modified with 32P.; Investigation was conducted into applying this to high density oligonucleotide arrays. Fluorophore-labelled oligonucleatide probes were covalently attached to silanized glass slides via a disulfide linkage. The slides were treated to fluorophore-labelled targets of complementary and non-complementary sequences and λ-exonuclease digestion. The slides ere scanned using a confocal microscope to verify these interactions. It was shown that this method of regeneration could be applied to microarray technology.