The Investigation of DNA and RNA Structural Differences Using Ultra High Performance Liquid Chromatography

DNA and RNA chromatography is extensively used for nucleic acid analysis. To better understand the chromatographic mechanisms by which DNA and RNA oligonucleotides are separated, ion pair reverse-pair ultra-high performance liquid chromatography (IP RP UHPLC) methods were developed. 11mer and 12mer DNA and RNA oligonucleotides of various compositions were used during this study. The first part of this study analyzed 11mer DNA and RNA oligonucleotides to better understand the chromatographic separations of DNA and RNA. The results gathered through the IP RP UHPLC analysis of these oligonucleotides demonstrated the existence of structural features that affect the chromatographic separations of DNA and RNA. This led to the IP RP UHPLC analysis of DNA and RNA oligonucleotides, of equal length and sequence, which either formed a 4 base-pair or 2 base-pair tetraloop secondary structure. The purpose of this investigation is to improve the isolation and purification of nucleic acid mixtures by understanding how DNA and RNA oligonucleotides interact with the stationary support but to also illuminate the role of structural features in nucleic acid separations. The characterization and the separation of the DNA and RNA oligonucleotides were achieved through a variety of methods including temperature melting experiments. The results gathered demonstrated the effectiveness of IP RP UHPLC to analyze the differences between DNA and RNA oligonucleotide separations. The DNA oligonucleotides eluted earlier than the RNA oligonucleotides which demonstrated that RNA has a different chromatographic mechanism than DNA. Differences between nucleic acid separations of fragments with the 2 base-pair tetraloop and 4 base-pair tetraloop structural modifications were also observed. The oligonucleotides with the 4 base-pair tetraloop eluted later than the oligonucleotides with the 2 base-pair tetraloop demonstrating the influence of structural modifications on the separation mechanisms of nucleic acids. The temperature melting experiments performed also confirmed that structural modifications influence the interaction between nucleic acids and stationary support. These results demonstrate the effectiveness of IP RP UHPLC to observe structural differences between DNA and RNA and as an alternative method to traditional methods, such as gel electrophoresis, to analyze oligonucleotides.