Structural changes of oxidized guanine-rich genomic regions

Aleksič, Simon (author), Plavec, Janez (mentor)

Guanine-rich regions in the genome play crucial roles in regulating gene expression and maintaining chromosomal stability, but are susceptible to oxidative damage, particularly at guanine residues. In addition, guanine-rich sequences can associate into G-quadruplexes, non-canonical structures that can act as steric hindrances in key cellular processes. Both processes are implicated in the onset of many diseases. In this dissertation, we investigated the structural changes induced by the oxidation of G-rich sequences. We focused on the incorporation of oxidative lesions and their effects on G-quadruplex formation and the structural equilibrium between double-stranded DNA and G-quadruplexes. Employing a combination of NMR, UV and CD spectroscopy, MD and DFT optimization, the work provides a comprehensive analysis of the structural and thermodynamic properties of oxidized G-rich sequences alone and in combination with a complementary C-rich strand. We synthesized and analyzed a series of oligodeoxyribonucleotides with strategically positioned 8-oxoguanine residues. NMR spectroscopy revealed that 8-oxoguanine residues form stable quartets within G-quadruplex cores, characterized by a unique hydrogen-bonding scheme. These quartets exhibit a larger central cavity compared to canonical G-quartets, confirmed by DFT-optimized models and cation localization studies. In two-stranded constructs, the position of 8-oxoguanine and abasic residues modulates the equilibrium between double-stranded DNA and G-quadruplexes. Despite the ability of spare G-tracts to rescue damaged sequences and facilitate G-quadruplex folding, most observed G-quadruplexes are kinetically trapped states, with double-stranded DNA as the preferred equilibrium structure. Constructs containing oxidative lesions exhibited varying degrees of structural polymorphism, influenced by lesion position. This work provides novel insights into the structural and functional consequences of oxidative damage in G-rich genomic regions. The findings underscore the significance of oxidized lesions in influencing DNA stability and G-quadruplex formation. These results have implications for understanding the molecular basis of oxidative stress, mutagenesis, and the potential targeting of G-quadruplex structures in therapeutic strategies.

• Language: English
• Keywords: genome, nucleic acids, guanine, G-quadruplex, DNA, NMR spectroscopy, UV spectroscopy, CD spectroscopy, structure determination
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-171734
• COBISS.SI-ID: 253247235
• Publication date in RUL: 01.09.2025

Secondary language

• Language: Slovenian
• Title: Strukturne spremembe oksidiranih z gvaninom bogatih regij genoma

Abstract

Z gvanini bogata območja v genomu imajo ključno vlogo pri uravnavanju izražanja genov in ohranjanju kromosomske stabilnosti, vendar so dovzetna za oksidativne poškodbe, zlasti na gvaninskih ostankih. Poleg tega lahko zaporedja, bogata z gvanini, tvorijo G-kvadruplekse, nekanonične terciarne strukture, ki lahko delujejo kot sterične ovire pri ključnih celičnih procesih. Oba procesa sta implicirana kot vzroka za nastanek številnih bolezni. V disertaciji smo raziskali strukturne spremembe, ki jih povzroči oksidacija zaporedij, bogatih z gvanini. Osredotočili smo se na vključevanje oksidativno poškodovanih nukleotidnih ostankov in njihov vpliv na tvorbo G-kvadrupleksov ter strukturno ravnotežje med dvoverižno DNK in G-kvadrupleksi. Z uporabo NMR, UV in CD spektroskopije, simulacij MD in optimizacije DFT delo zagotavlja celovito analizo strukturnih in termodinamičnih lastnosti oksidiranih zaporedij, bogatih z gvanini, tako samostojno kot v kombinaciji s komplementarno verigo, bogato s citozini. Sintetizirali in analizirali smo vrsto oligodeoksiribonukleotidov z vgrajenimi 8-oksogvaninskimi ostanki. Uporaba metod NMR spektroskopije pokazala, da 8-oksogvaninski ostanki tvorijo stabilne kvartete v jedru G-kvadrupleksa, za katere je značilna edinstvena shema vodikovih vezi. Ti kvarteti imajo v primerjavi s kanoničnimi G-kvarteti bistveno večjo osrednjo votlino, kar potrjujejo DFT-optimizirani modeli in študije lokalizacije kationov. V dvoverižnih konstruktih z verigama, bogatima z gvanini in citozini, položaj 8-oksogvaninskega in abazičnega ostanka spreminja ravnotežje med dvoverižno DNK in G-kvadrupleksi. Kljub sposobnosti dodatnih G-traktov, da rešujejo poškodovana zaporedja in olajšajo tvorbo G-kvadrupleksa, je večina opaženih G-kvadrupleksov kinetično ujetih, pri čemer je ravnotežna struktura dvoverižna DNA. Konstrukti, ki so vsebovali oksidativne poškodbe, so izkazovali različne stopnje strukturnega polimorfizma, na katerega je vplival položaj poškodbe. To delo zagotavlja nov vpogled v strukturne in funkcionalne posledice oksidativnih poškodb v genomskih regijah, bogatih z gvanini. Ugotovitve poudarjajo pomen oksidativnih poškodb pri vplivanju na stabilnost DNK in tvorbo G-kvadrupleksov. Ti rezultati vplivajo na razumevanje molekularne osnove oksidativnega stresa, mutageneze in morebitnega ciljanja oksidativno poškodovanih G-kvadrupleksov v terapevtske namene.

• Keywords: genom, nukleinske kisline, gvanin, G-kvadrupleksi, DNA, NMR spektroskopija, UV spektroskopija, CD spektroskopija, določanje strukture