G-Triple Chemiluminescent DNAZyme Based Biosensor for the Detection of Calcium(II) Ions in Solution and the Biological Functionality of Kinase Enzymes and the Rationale for their Employment as Promising Drug Targets, Substrate Specific Inhibition of P38alpha

G-quadruplex DNA structures, which are formed from planar stacks of G-tetrads and follow Hoogsteen hydrogen bonding, have recently received attention as recognition elements in bioanalytical assays. DNAzymes are functionally active, synthetic single-stranded DNA molecules that are capable of catalyzing biological reactions similarly to proteins or RNAzymes. The majority of DNAzymes are synthesized by an in vitro selection method known as systematic evolution of ligands by exponential enrichment (SELEX) and can catalyze a wide variety of reactions, including the cleavage or ligation of nucleic acids, RNA branching, phosphorylation, synthesis of nucleopeptide bonds, porphyrin metalation of DNA. One class of DNAzymes mimics peroxidase by catalyzing the oxidation of luminol or 2,2’-azino-bis(3ethylbenzthiazoline6-sulphonic acid (ABTS) in the presence of H2O2. These peroxidase-mimicking DNAzymes consist of G-quadruplex structures, which provide a suitable hydrophobic hemin binding pocket that contributes to oxidation enhancement. G-triplex DNA structures are characterized by G:G:G triads and are stabilized by Hoogsteen hydrogen bonding. Originally identified as folding intermediates for G-quadruplexes, certain truncated G-quadruplex forming sequences have recently been shown to form stable G-triplex structures, although the “rules” for which sequences fold into stable G-triplexes are not well defined. While G-quadruplex-based peroxidase mimicking DNAzymes have been employed in a number of sensor designs, there have been no examples of chemiluminescence-based DNAzymes based on G-triplex motifs. We hypothesized that the G-triplex could function as a type of peroxidase mimicking DNAzyme that could have some advantages in sensor design. Here we have developed a DNAzyme based assay which shows that a variety of previously described and newly reported G-triplexes are able to catalyze the oxidation of luminol to produce chemiluminescence. Furthermore, various G-triplex forming sequences can be used to design label-free G-triplex based biosensors for the detection of Ca2+ ions. To produce quick and reliable assays, we also focused on the optimization of an Opentrons liquid handling robot which would provide assay results with the lowest standard deviation error. A simple and cost-effective assay such as this would be beneficial to the diagnostic field for the current techniques of Ca2+ ion detection requires sophisticated instrumentation and complicated sample preparation. Utilizing the results from the optimization of the Opentrons liquid handling robot, we have also developed a miniaturized assay format suitable for protein kinase inhibition by utilizing the ADP-Glo kit from Promega. We first started by validating a protein kinase inhibition assay using Protein Kinase A (PKA) and the pan-kinase inhibitor Staurosporine with the final goal of evaluating the ability of rooperol analogues to inhibit the protein kinase p38a. p38a is a Mitogen Activated Protein Kinase (MAPK) which when dysregulated can cause diseases such as autoimmune disorders, cardiovascular disease, inflammation, and cancer. To date, no p38a kinase inhibitor has been developed with a high level of specificity due to targeting the ATP binding site with is highly conserved among all protein kinases. This consequence is seen in clinical trials, where the drug will decrease the disease severity for patients for only a few months. Previously, it has been determined that rooperol, a polyphenolic compound, is able to inhibit the activity of p38a by binding to the docking recruitment site (DRS) instead of the ATP binding site. The DRS of MAPK p38a recognizes specific substrates thus DRS targeted inhibition may display substrate selective inhibition which would dramatically increase the specificity of the inhibitor. However, it has been shown that rooperol undergoes extensive metabolism and has a short half-life in human. Because of this reason, it is essential to obtain rooperol analogues and determine if they are effective at inhibiting p38a. In this project, we have developed a G-triplex based DNAzyme chemiluminescent biosensor for the detection of calcium(II) ions which would serve a practical use for residential areas, businesses, and laboratories. Moreover, utilizing an ADP-Glo assay obtained from Promega, we have attempted to inhibit PKA using the pan-kinase inhibitor staurosporine with the future goal of inhibiting p38a via rooperol analogues.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Biochemistry

Grantor

Texas State University

Year dc:date.issued

2023

Author dc:creator

• Williams, Elizabeth

Advisor dc:contributor.advisor

• Kerwin, Sean Michael

Committee members dc:contributor.committeemember

• Lewis, Karen A.
• Peterson, Ryan

• G-triplex
• DNAzyme
• biosensor
• p38-alpha
• MAPK
• rooperol
• inhibition

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10877/20237

OAI identifier oai:identifier

oai:digital.library.txst.edu:10877/20237