Morpholino-dna interaction study on morpholino microarray

Although technologies based on surface hybridization to DNA monolayers continue to be widely and successfully used, their performance is constrained by the fundamental properties of nucleic acids, including their high degree of charging. As an alternative approach, this thesis considers a type of a non-ionic DNA analogue, morpholino (MO), for such applications. Under low ionic strengths the neutral backbone of MOs provides higher affinity binding to complementary DNA than native DNA-DNA hybridization. This feature helps select out MO-DNA surface hybridization while destabilizing solution DNA-DNA interactions and secondary structure in the analyte. This thesis also addresses key considerations pertaining to fabrication and demonstrative applications of MO microarrays, as well as analyzes fundamental aspects of the thermodynamics of MO-DNA hybridization on solid supports. A total-internal-reflection fluorescence (TIRF) imaging approach is introduced for determining surface melting transitions of MO-DNA duplexes, from which thermodynamic data are derived. From an application perspective, optimal resolution of competing design goals is addressed, for example in striking a compromise between destabilization of sample secondary structure in solution while retaining sufficient surface hybridization affinity to provide strong detection signals. Moreover, a fluorescence un-quenching method is introduced for MO microarrays as a facile way of detecting unlabeled analyte, and a small scale study of gene expression levels in Legionella pneumophila is demonstrated.