Development and utilization of spectroscopic and chemical methodologies for investigating pKa shifting in nucleic acids

Open Access
Wilcox, Jennifer Lynn
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
July 31, 2013
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Squire J Booker, Committee Member
  • Scott A Showalter, Committee Member
  • Andrey S Krasilnikov, Committee Member
  • RNA
  • nucleic acids
  • 2-aminopurine fluorescence
  • pKa shifting
RNA has historically been thought to play an intermediary role in the flow of genetic information between DNA and proteins. With its 2’ hydroxyl, RNA is much less stable than DNA, which makes it amenable to a transitional role rather than one of storage. Proteins are equipped with 20 chemically diverse amino acid side chains, surpassing the functional potential of the four heterocycles that make up RNA. However, with the discovery of catalytic RNA (or ribozymes) in 1982, RNA was no longer limited to the transitional role it was once believed to have. The discovery of catalytic RNA shed light on RNA’s potential to actively participate in biological processes. Proteins can act catalytically as a result of an electrostatic contribution with pKa’s of amino acid side chains far from neutrality to form a charged species at neutrality or with side chain pKa’s near neutrality to allow for participation through proton transfer. However, the pKa’s of single stranded nucleobases are far from neutrality, inhibiting participation in catalysis. Because Watson-Crick base pairing requires the formation of hydrogen bonds, the pKa’s of the nucleobases are shifted even further from neutrality, significantly decreasing the potential for catalytic activity. The pKa’s of the nucleobases must be shifted towards neutrality in order to act catalytically in a manner analogous to amino acid side chains. Some non-canonical interactions shift RNA pKa’s towards neutrality, enabling the nucleobases to act catalytically. Interactions that facilitate protonation of nucleobases occur in both secondary and tertiary motifs and allow RNA to play a much more diverse role than the intermediary role once thought. Protonated nucleobases participate in many biological processes such as general acid-base catalysis of ribozymes, programmed ribosomal frameshifting, miRNA processing, and RNA editing. We hypothesize that RNA systems can contain protonated motifs that participate in biological processes but confirmation of protonation in these systems is limited by existing methods. To determine the driving forces for pKa shifting, the development of a new method for determining pKa’s was essential. Spectroscopic methods currently exist to quantify pKa shifting in nucleic acid systems; however, spectroscopic analysis by these methods requires high sample concentration, costly sample preparation and lengthy experimental time. Additionally, some existing spectroscopic methods such as UV-Vis spectroscopy and CD require a large conformational change to produce an observable signal change between protonated and deprotonated states, therefore limiting the size of systems that can be analyzed. I developed a new method using fluorescence spectroscopy to determine pKa’s in nucleic acids. Development of this method utilized fluorescence quenching of 2-aminopurine (2AP), a fluorescent isomer of adenine, when stacked. Positioning the 2AP adjacent to the protonated base allowed for high fluorescence when the base pair wasn’t formed and low fluorescence when the base pair was formed and the 2AP fluorescence was quenched due to stacking. The pKa’s obtained with fluorescence spectroscopy agreed with those obtained with a previously developed 31P NMR method. The pKa of adenine in an A+C wobble in a DNA hairpin was measured accurately with fluorescence at one-third of the cost and 10 percent of the required experimental time of 31P NMR. In addition to utilization of the technique on DNA secondary motifs, I also determined the pKa of a cytidine participating in a base quartet in the beet western yellows virus (BWYV) RNA. Protonation of C8 in the BWYV RNA enables pseudoknot formation and programmed ribosomal frameshifting. Previous studies have been performed in the Bevilacqua lab to determine the salt, temperature, and context dependence of pKa shifting in DNA while the sequence remained constant. In nature, however, sequence can also contribute to pKa shifting. Using 31P NMR, I determined the pKa of adenine in an A+C wobble in an RNA hairpin in various sequence and structural environments. Strengthening the base pairing of nearest neighbors from two weak A-U nearest neighbors to two strong G-C nearest neighbors enabled an additional pKa shift of 1.6 units, up to 8.1. A similar dependence on the melting temperature and free energy on the oligonucleotide sequence was found through thermal denaturation experiments as both parameters were more favorable with increased nearest neighbor base pairing strength, indicating that the addition of stability and more favorable interactions contributes to the stability of the construct as well as pKa shifting. The pKa’s determined were between 6.5 and 8.1, suggesting that sequence could play a role in tuning pKa’s near physiological pH. We hope that the trends presented in this study will serve as a model to aid in the determination of the roles protonated bases play in various dsRNA-mediated processes in biology. The aforementioned studies use fluorescence and NMR spectroscopies to determine pKa’s in nucleic acids. In those cases, systems were designed with a suspected protonated nucleobase whose identity was known before quantifying pKa shifting. Although spectroscopic methods are useful in determining the pKa of nucleobases with elevated pKa’s to confirm protonation and quantify pKa values, many systems contain protonated nucleobases of unknown identity and spectroscopic methods are unable to readily identify the protonated nucleobase. In an effort to address this problem, I made significant progress towards the development of a chemical methodology to damage and map protonated bases in large RNA systems utilizing elevated reactivity of cationic over neutral nucleobases. Ultimately, the development of a reagent to map protonated bases in large RNA systems should be possible but significant efforts need to be made in the future to determine the optimal reaction and analysis conditions.