Characterization of thermodynamically stabilizing mutations in a light-producing deoxyribozyme using NMR spectroscopy

• Thesis title in Czech: Charakterizace termodynamicky stabilizujících mutací ve světlo-produkujícím deoxyribozymu pomocí NMR spektroskopie
• Thesis title in English: Characterization of thermodynamically stabilizing mutations in a light-producing deoxyribozyme using NMR spectroscopy
• Key words: deoxyribozym, katalytická DNA, termostabilita, tání, nukleární magnetická rezonance
• English key words: deoxyribozyme, catalytic DNA, thermostability, melting, nuclear magnetic resonance
• Academic year of topic announcement: 2020/2021
• Thesis type: Bachelor's thesis
• Thesis language: angličtina
• Department: Laboratory of NMR Spectroscopy (31-204)
• Supervisor: doc. RNDr. Zdeněk Tošner, Ph.D.
• Author: hidden - assigned by the advisor, waiting for guarantor's approval
• Date of registration: 10.11.2020
• Date of assignment: 10.11.2020
• Date of electronic submission: 23.08.2021
• Date of proceeded defence: 07.09.2021
• Opponents: Mgr. Klára Kohoutová
• Advisors: Edward Arthur Curtis, Ph.D.

Preliminary scope of work

Ve spolupráci se skupinou Dr. Curtise se budeme věnovat studiu světlo-produkujícímu DNA enzymu a jeho mutacím, jejichž stabilita vzhledem k teplotě tání bude sledována pomocí 1H NMR spektroskopie.
Tato práce bude vedena v anglickém jazyce. Bližší náplň práce je tedy uvedena v anglické verzi.

Preliminary scope of work in English

Like proteins, DNA molecules can catalyze chemical reactions. Inspired by the potential uses of catalytic DNA, an artificial evolution was used in Dr. Curtis’ lab to identify a deoxyribozyme that catalyzes a chemiluminescent reaction. Our current goal is to obtain a structure of this deoxyribozyme at atomic-level resolution.
In the case of both protein and nucleic acid enzymes, high-resolution structural characterization is often facilitated by mutations that increase stability at high temperatures. A rational design approach was chosen to identify such modifications; a series of previously described stabilizing loops were inserted into a hairpin in the catalytic core of the deoxyribozyme, and NMR spectroscopy was used to characterize the effect of these loop sequences on the melting temperature of the deoxyribozyme.
Variable-temperature (VT) ¹H-NMR spectra of these variants were measured, and the melting transition of their structure was investigated. In addition, the ability of deoxyribozymes to retain their catalytic activity at higher temperatures was studied and provided further insight into the sequence elements that demonstrate a thermostabilizing effect.