Leveraging Unnatural Cytosine Analogs and Engineered DNA Modifying Enzymes to Parse Natural Cytosine States in DNA Christian E Loo

Format:

Book

Thesis/Dissertation

Author/Creator:

Loo, Christian E., author.

Contributor:

University of Pennsylvania. Biochemistry and Molecular Biophysics., degree granting institution.

Language:

English

Subjects (All):

0307.

0369.

0487.

Local Subjects:

0307.

0369.

0487.

Physical Description:

1 electronic resource (195 pages)

Contained In:

Dissertations Abstracts International 87-07B

Place of Publication:

Ann Arbor : ProQuest Dissertations and Theses, 2025

Language Note:

English

Summary:

Ordered, chemically linked nucleotides both underlie the code for life and explain the genesis of diversity in organisms. As an added layer above A, C, G, and T, purposeful chemical modifications, such as methylation at the 5-position of cytosine (5mC), are introduced to the nucleobases by enzymes like DNA methyltransferases (MTase). These DNA modifications expand the coding capacity of DNA and can serve as key epigenetic marks that dictate gene expression and cellular identity. Thus, understanding where and how these marks act is critical for deciphering their role in biology and disease. However, existing methods for detecting and mapping cytosine modifications require trade-offs between accessibility, resolution, accuracy, and information content. In this thesis, I address these challenges by leveraging unnatural cytosine analogs alongside engineered DNA modifying enzymes that function selectively as either 'writers' to mark DNA or as 'readers' to detect modified states.I begin by elucidating the mechanistic basis of an engineered MTase, M.MpeI N374K, which deposits an unnatural DNA modification. Structural and biochemical analyses reveal the key active site interactions involved. Notably, these residues are highly conserved across MTases, suggesting that this bio-orthogonal labeling strategy may have broader applicability for mapping epigenetic states.To improve global quantification of DNA modifications, I develop Sparse-Sequencing, a shallow-depth sequencing method that enables efficient detection of rare modifications. Although Sparse-Seq is compatible with various sequencing modalities, I further refine the approach by introducing bACE-Seq. This method employs novel dually resistant sequencing adaptors to enable simultaneous and efficient tracking of 5mC and its TET-oxidized product, 5-hydroxymethylcytosine (5hmC). Applying Sparse-bACE-Seq to developing mouse brains reveals context-dependent epigenetic dynamics.To advance the integration of genetic and epigenetic analyses, I introduce Integrated-Sequencing. This method employs a novel DNA deaminase-helicase fusion 'reader' enzyme that selectively discriminates epigenetic cytosine states, while maintaining genetic information within a synthetic copy strand embedded with unnatural cytosine analogs.Together, these innovations provide a suite of purpose-built tools for high-accuracy DNA modification mapping, significantly enhancing our ability to interrogate the functional landscape of the genome with unprecedented precision

Notes:

Advisors: Kohli, Rahul M. Committee members: Black, Ben E.; Sellmyer, Mark A.; Wu, Hao; Zhou, Wanding

Source: Dissertations Abstracts International, Volume: 87-07, Section: B.

Ph.D. University of Pennsylvania 2025

Vendor supplied data

Local Notes:

School code: 0175

ISBN:

9798276005584

Access Restriction:

Restricted for use by site license