DESIGN OF HIGH-YIELDING NON-ENZYMATIC DNA AND RNA LIGATION SYSTEMS

The demonstration of enzyme-free replication of nucleic acids, in particular RNA, will lend credence to the RNA world hypothesis which states that RNA was the first polymer on the prebiotic earth. One major challenge that currently still exists is how substrates can undergo ligation (i.e. form a covalent bond) in the absence of enzymes. The use of activated species such as imidazole and water soluble carbodiimides have been proposed as means for ligation. However, obtaining the activating agents by a prebiotic route is an active area of research with no consensus yet on an exact pathway. Cyclic phosphate intermediates offer a potential route to prebiotic ligation of RNA substrates, but these reactions are often marred by low yields. In order to demonstrate true enzyme-free replication, a prebiotically plausible route to non-enzymatic ligation must be developed. In this Thesis, a systematic non-enzymatic ligation method is described for both DNA and RNA ligating systems. DNA is used as a model system to investigate the limits of non-enzymatic ligation in a system that is prone to high yields, unlike the RNA cyclic phosphate system. This Thesis demonstrates how several factors such as binding of the substrates to the templates, side product reactions, and stacking of the bases at the ligation site can affect yields in DNA ligation. Applying these findings to the RNA cyclic phosphate ligation system, the role of sequences and base-pairing at the ligation nick, in addition to cyclic phosphate hydrolysis, were emphasized as potential barriers for attaining high yielding RNA systems in a prebiotic world.

Level thesis:degree_level

Doctoral

Department dc:contributor.department

Chemical and Biomolecular Engineering

Grantor dc:publisher

Georgia Institute of Technology

Year dc:date.issued

2021

Author dc:creator

• Obianyor, Chiamaka

Advisors dc:contributor.advisor

• Hud, Nicholas V.
• Grover, Martha A.

Committee members dc:contributor.committeemember

• Paravastu, Anant
• Wilson, Corey
• Styczynski, Mark

• Nucleic acids
• Chemical ligation

Handle dc:identifier.uri

http://hdl.handle.net/1853/64748

OAI identifier oai:identifier

oai:repository.gatech.edu:1853/64748