Date of Award




Document Type


Degree Name

Doctor of Philosophy (PhD)


Department of Chemistry

Content Description

1 online resource (xv, 194 pages) : illustrations (some color)

Dissertation/Thesis Chair

Maksim Royzen

Committee Members

Gabriele Fuchs, Jia Sheng, Mehmet Yigit


Oligonucleotides, CRISPR (Genetics), Biomedical engineering, Diels-Alder reaction

Subject Categories

Biochemistry | Biology | Chemistry


This thesis describes the synthesis of an oligonucleotide containing an unnatural neutralbackbone. The backbone was constructed using bio-orthogonal inverse electron demand Diels- Alder chemistry between trans-cyclooctene (TCO) and tetrazine (Tz). A TCO phosphoramidite was synthesized and attached to solid support for strand propagation. Uridine monomers containing either bis-TCO or bis-Tz were also synthesized. The oligonucleotide strand was formed by sequential ligation of the bis-Tz and bis-TCO monomers. The oligonucleotide was analyzed using mass spectrometry. This thesis also describes the synthesis of sgRNAs containing non-canonical nucleobases, m1A, m6A, s2U and s4U using the ‘split-and-click’ methodology. The synthetic sgRNAs were assembled using shorter (~30-nt) RNA strands functionalized with Tz and longer RNA strands (~70-nt) functionalized with TCO. These RNA strands were ligated using bio-orthogonal inverse electron demand Diels-Alder chemistry between TCO and Tz. 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. sgRNAs containing non-canonical nucleobases, m1A, m6A, s2U and s4U were synthesized and evaluated for their ability to facilitate nuclease activity.