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Full text of this paper is not available in the ETD Center. Copies may be available for inter-library loan from Kent State University or may be available for purchase from Proquest/UMI

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Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity

Schmocker, Stefani P
http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092

Abstract Details

2020, BS, Kent State University, College of Arts and Sciences / Department of Chemistry and Biochemistry.
The methylation of the sixth position of adenine (m6A) is an epitranscriptomic modification that has been found to play a vital role in early cell differentiation and embryonic development. The removal of the m6A modification is facilitated by the "eraser" protein FTO. The protein FTO (fat mass and obesity-associated protein) is a non-heme Fe2+ and α-ketoglutarate-dependent dioxygenase demethylase enzyme whose activity has been recently linked to spermatogenesis and cancer progression. However, little is known about the biophysical nature of its binding to its RNA targets. First, FTO was produced in BL21 (DE3) Escherichia coli transformed with the PET45-C+1 plasmid and purified using affinity chromatography. Thin Layer Chromatography with32 P-labeled, m6A-containing target RNA which was incubated with FTO resulted in loss of the m6A modification, as indicated by changes in the spotting pattern compared to a control reaction. Additionally, FRET experiments were used to determine the effect of FTO's cofactors (α-ketoglutarate and Fe2+) on its binding thermodynamics, revealing slightly increased dissociation constant (KD) values in the absence of each cofactor (α-ketoglutarate and Ni2+, an Fe2+ analog). This suggests that FTO binding to m6A RNA is slightly strengthened when both cofactors are present in solution. FRET experiments also indicated the potential of a phage-display isolated peptide to serve as an inhibitor of FTO's demethylase activity.
Sanjaya Abeysirigunawardena, PhD (Advisor)
Soumitra Basu, PhD (Committee Member)
Srinivasan Vijayaraghavan, PhD (Committee Member)
Timothy Meyers, PhD (Committee Member)
88 p.
Biochemistry
FRET; FTO; TLC; N6-methyladenosine; demethylase

Recommended Citations

Citations

  • Schmocker, S. P. (2020). Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity [Undergraduate thesis, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092

    APA Style (7th edition)

  • Schmocker, Stefani. Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity. 2020. Kent State University, Undergraduate thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092.

    MLA Style (8th edition)

  • Schmocker, Stefani. "Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity." Undergraduate thesis, Kent State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092

    Chicago Manual of Style (17th edition)

ksuhonors1588272250950092