DNA aptamers: A novel approach to chemical separations

Aptamers are short, generally single stranded, DNA or RNA oligomers, which form specific three-dimensional structures in solution and are isolated by combinatorial methods to bind to specific organic molecules or macromolecules with high binding affinities. The interest in aptamers to date has focused primarily on their use in pharmaceutical and medicinal sciences. For example, the thrombin binding DNA aptamer has been studied mainly for its inhibitory activity towards thrombin for prevention of blood coagulation. A few articles recently have discussed analytical applications of aptamers, exploring them mainly for isolation and sensing of target molecules.; Our interest in aptamers lies in studying their interactions with non-target compounds towards development of aptamers as stationary phase reagents for chemical separation of non-target compounds. This research project has involved three main stages. The first stage involved the study of aptamer conformation and its stability under various solvent conditions such as absence and presence of KCl, and presence of organic modifiers such as methanol and acetonitrile. These were performed using absorbance and circular dichroism techniques.; The second stage involved the study of binding interactions of the thrombin binding aptamer and other related sequences with non-target compounds by spectroscopic techniques such as UV-Vis absorption, circular dichroism, fluorescence emission, anisotropy and fluorescence lifetime. The differences observed among the interactions of the aptamer with different compounds within the same class formed the basis for developing the aptamers as stationary phases for separation of these compounds. These batch mode studies revealed information regarding the molecular basis for these interactions. These results served to justify the technique and rationale behind the hypothesis that separations of non-target compounds can be achieved using aptamers.; The final stage of this project involved the attachment of aptamers to a capillary surface using various linker chemistries and perform separations of the non-target compounds by capillary electrochromatography. The attachment experiments were performed by a colleague, Lijuan Li, after much discussion and modification of the original published methods. These linkers for aptamer attachment were investigated for stability under separation conditions and for surface coverage based on performance. Separations of non-target compounds were then attempted by capillary electrochromatography.; CEC separations of various binary mixtures of PAHs (naphthalene-phenanthrene, naphthalene-benzo[a]pyrene and naphthalene-benzo[ghi]perylene), amino acids (D-tryptophan-D-tyrosine and L-proline-L-arginine) and amino acid enantiomers (D and L tryptophan) were performed. An attempt was made to separate the mixture of 17 L-amino acids and also 16 EPA priority pollutant PAHs. For PAHs, both absorbance detection using the in-built UV-detector and fluorescence detection using CE-MHF interface was performed. Various parameters were studied in order to optimize the separation. Complete separation of some of these compounds was achieved, while only partial resolution was obtained for others. Control experiments were performed in order to establish that the separations achieved were in fact a result of aptamer interaction with the analyte.; The variety of structures offered by aptamers, combined with the numerous modes of interaction of single-stranded DNA with a whole range of chemical species, suggest that the possibilities for applications of aptamer stationary phases are enormous.