Vpliv terminalnih koncev in narave kationov na tvorbo G-kvadrupleksov

Pavc, Daša (author), Šket, Primož (mentor)

Preučevali smo vpliv terminalnih koncev oligonukleotidov in narave prisotnih kationov na zvijanje in multimerizacijo G-kvadrupleksov do dolgih nanostruktur, G-žičk. Uporaba terminalnih GC koncev oligonukleotidov lahko vodi do multimerizacije G-kvadrupleksov preko prepletanja in do nastanka daljših nanostruktur. Vpliv prisotnosti 15NH4+ in K+ ionov smo preučevali na G-kvadrupleksih, katere tvorita oligonukleotida d(GCG2AG4AG2) in d(GCG2AG4AG2CG) poimenovana GCn in GCnCG. Pokazali smo, da prisotnost 15NH4+ ali K+ ionov sproži multimerizacijo preko nalaganja 3'-terminalnih G-kvartetov GCn G-kvadrupleksa, ki pa jo 3'-GC konci preprečijo v primeru GCnCG G-kvadrupleksa. V 3'-3' naloženem GCn G-kvadrupleksnem multimeru smo opazili pet vezanih 15NH4+ ionov, med katerimi se eden nahaja tudi na 3'-3' medkvadrupleksni površini. Za 15NH4+ ione vezane v 3'-3' naloženem GCn G-kvadrupleksnem multimeru smo opazili počasno dinamiko izmenjave. Nasprotno, prisotnost 3'-GC konca pospeši izmenjavo vezanih 15NH4+ ionov med vezavnimi mesti v GCnCG G-kvadrupleksu in 15NH4+ ioni v raztopini. 15NH4+ ioni znotraj GCnCG G-kvadrupleksa se premikajo samo v eno smer, kar je lastnost ionskega kanala. Pokazali smo, da v prisotnosti K+ ionov oligonukleotid d(G2AG4AG2) multimerizira do G-žičk. S spreminjanjem pogojev v raztopini in načina priprave vzorca smo našli pet G-kvadrupleksnih struktur, ki nastanejo tekom samozdruževanja d(G2AG4AG2) do G-žičk. Z uporabo NMR spektroskopije smo določili topologije zvitja omenjenih petih G-kvadrupleksnih struktur in tako dobili vpogled v mehanizem nastanka G-žičk na molekularnem nivoju. Spreminjanje nukleotidov v zankah nam je omogočilo spreminjanje lastnosti G-žičk, s simulacijami MD pa smo razložili kako nukleotidi v zankah vplivajo na dolžino nastalih G-žičk. Preučevali smo tudi možnosti nastanka G-kvadrupleksnih struktur višjega reda v biološkem kontekstu na primeru oligonukleotida iz človeškega telomernega zaporedja, ki vsebuje pet G-traktov, d(TAG3(T2AG3)4). Ugotovili smo, da prisotnost petega G-trakta povzroči nastanek paralelnega G-kvadrupleksa s T2AG3 prostim koncem. Multimerizacija je bolj verjetna pri paralelnih, kot pri hibridnih G-kvadrupleksih, kjer robne zanke ovirajo nalaganje preko terminalnih G-kvartetov.

• Language: Slovenian
• Keywords: G-kvadrupleksi, multimerizacija, strukture višjega reda, G-žičke, samozdruževanje, NMR, DNA nanotehnologija
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-137362
• COBISS.SI-ID: 115484419
• Publication date in RUL: 14.06.2022

Secondary language

• Language: English
• Title: Influence of terminal ends and nature of cations on G-quadruplex formation

Abstract

We studied how oligonucleotides’ terminal ends and nature of present cations influence formation and multimerization of G-quadruplexes to long nanostructures, G-wires. Terminal GC ends in oligonucleotide might promote G-quadruplex multimerization via interlocking and thus formation of longer nanostructures. The effect of the presence of 15NH4+ and K+ ions was studied on G-quadruplexes formed by oligonucleotides d(GCG2AG4AG2) and d(GCG2AG4AG2CG), named GCn and GCnCG. We showed that the presence of 15NH4+ or K+ ions induces multimerization via stacking of 3’-terminal G-quartets in GCn G-quadruplex, which is precluded by 3’-GC ends in the case of GCnCG G-quadruplex. We observed five 15NH4+ions bound in 3’-3’ stacked GCn G-quadruplex multimer, with one located at 3’-3’ stacking interface. 15NH4+ ions bound within 3’-3’ stacked GCn G-quadruplex multimer exhibit slow exchange dynamics. Contrary, presence of 3’-GC ends accelerates exchange ofbound 15NH4+ ions between binding sites in GCnCG G-quadruplex and with 15NH4+ ions in bulk solution. 15NH4+ ions within GCnCG G-quadruplex show unidirectional movement, which is characteristic for ion channels. We showed that in the presence of K+ ions, d(G2AG4AG2) self-assembles into G-wires. By varying solution conditions and sample preparation procedure, we found five G-quadruplex structures, which are formed in d(G2AG4AG2) G-wire self-assembly. Using NMR spectroscopy we determined folding topologies of mentioned five G-quadruplex structures and thus obtained insight into mechanism of G-wire self-assembly on molecular level. Changing the nucleotides in loops enabled us to manipulate G-wires’ properties. MD simulations provided rationale on how nucleotides in loops influence length of formed G-wires. We also studied the possibility of higher-order G-quadruplex structure formation in biological context on oligonucleotide from human telomere region, containing five G-tracts, d(TAG3(T2AG3)4). We showed that the presence of additional G-tract leads to formation of parallel G-quadruplex with 3’-terminal T2AG3 overhang. Multimerization is more likely for parallel than hybrid G-quadruplexes, where lateral loops hinder stacking of terminal G-quartets.

• Keywords: G-quadruplexes, multimerization, higher-order structures, G-wires, self-assembly, NMR, DNA nanotechnology