The separation of hairpins and modified nucleic acids by ion pairing reversed-phase high performance liquid chromatography.

摘要

DNA and RNA are used in applications such as PCR, genotyping, microarrays, therapeutics, and structural studies. Many of these applications require high purity (≥99%) oligonucleotides to reduce the inclusion of undesirable species or reactions into the experiment. The development of efficient and economic purification schemes is thus a problem of critical significance.;Due to the ionic nature of oligonucleotides, separation methods are typically based on charge, and the realm of chromatographic separations of nucleic acids has been dominated by anion exchange and ion-pairing reversed-phase retention mechanisms. More recently, ion-pairing reversed-phase liquid chromatography (IP-RPLC) has gained notoriety for its applicability to replace gel methods for restriction digest analysis and mutation detection of double stranded DNA.;This dissertation research expands and probes the limits of IP-RPLC separation of nucleic acids. The separation of hairpins and modified sequences show that this chromatographic separation method is unique in its sensitivity to conformation and sequence. Model hairpins are used to examine the separation and purification of RNA constructs of the same length and charge, but different conformational stability. Deoxyoligonucleotides with small alkyl modifications present in the sequence are used to show that the identity of the base is extremely important to the retention of an oligonucleotide. And finally, investigation in the areas of nucleic acid scission and photo-product formation have provided the ground work for extending the utility of IP-RPLC further into the realm of non-traditional chromatographic analysis, such as monitoring product formation of nucleic acid cleavage, foot-printing, and the formation of oxidation products.;These three areas of work clearly show that IP-RPLC is not only a separation method that is based on charge or length of the nucleic acid. Characteristics such as conformation and sequence can be exploited to enhance the separation and purification of nucleic acids, making this technique more versatile and amenable to their analysis than other chromatographic and electrophoretic techniques.