Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Content Description

1 online resource (xix, 156 pages) : illustrations (chiefly color)

Dissertation/Thesis Chair

Maksim Royzen

Committee Members

Mehmet Yigit, Alexander Shekhtman, Jia Sheng, Gabriele Fuchs

Keywords

Bio-orthogonal chemistry, DNA hydrogel, Long RNAs, oligonucleotide synthesis and purification, Paramagnetic NMR, Oligonucleotides, Colloids, Magnetic resonance microscopy, Circular dichroism, Nucleic acids, Proteins

Subject Categories

Biochemistry | Chemistry | Nanoscience and Nanotechnology

Abstract

This thesis describes a chemical method for alternative oligonucleotide purification that is non-chromatographic and gel-free and allows to routinely synthesize and purify long functional RNA strands. The purification of long RNAs is based on the bio-orthogonal inverse electron demand Diels-Alder (IEDDA) chemistry between trans-cyclooctene (TCO) and tetrazine (Tz). Target oligonucleotide strands are selectively tagged with Tz and can be captured and purified from the failure sequences with immobilized TCO. RNA strands are synthesized on solid support through a photolabile linker to avoid the loss of Tz tag. Purity of the isolated oligonucleotides was evaluated using gel electrophoresis, HPLC and mass spectrometry, while functional fidelity of the sgRNA was confirmed using CRISPR-Cas9 experiments and flow cytometry.This thesis also describes a small-molecule induced DNA hydrogel which can be programmed to encapsulate and release therapeutic and fluorescent compounds. The hydrogel was formed by the assembly of “Y”-shaped DNA motif and polyA (A15) DNA mediated by a small molecule, cyanuric acid (CA), which was reported to promote the assembly of unmodified polyA strands into non-canonical motifs. Hydrogel formation was analyzed by circular dichroism (CD) spectroscopy. The proposed system displays comparable mechanical properties with low concentration of nucleic acid building blocks, confirmed by rheology study. Encapsulation of a variety of materials were characterized by fluorescence imaging and the release profile was studied. Lastly, we developed a method to introduce a lanthanide-based paramagnetic probe to investigate nucleic acid-protein interactions using paramagnetic 2D-NMR techniques. The probe contains highly paramagnetic Dy(III) or Eu(III) metals, coordinated with a macrocyclic ligand, TCMC, which was previously reported to eliminate the nuclease activity of lanthanide metals. We describe chemical synthesis of the probe and a general approach for its incorporation into a DNA of interest. A proof-of-principle study was conducted with a well-established viral RNA-protein complex using the HIV nucleocapsid protein 7 (NCp7) and HIV-1 viral RNA stem loop 3 (SL3). Distance-dependent paramagnetic resonance enhancement (PRE), as well as pseudo-contact chemical shift (PCS) effects have been observed during the in vitro HSQC NMR experiments. Based on these data a visual binding surface of NCp7-SL3 complex has been generated.

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