Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization

Given the increasing interest in i-motif structures, obtaining such structures as well as detailed structural information under physiological conditions have become hot topics in the structural biology field. In this context, the main objectives of this thesis are focused on the design and detailed...

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Author: Mir Morro, Bartomeu
Format: doctoral thesis
Status:Published version
Publication Date:2019
Country:España
Institution:CBUC, CESCA
Repository:TDR. Tesis Doctorales en Red
OAI Identifier:oai:www.tdx.cat:10803/668126
Online Access:http://hdl.handle.net/10803/668126
Access Level:Open access
Keyword:Química orgànica
Química orgánica
Organic chemistry
Àcids nucleics
Ácidos nucleicos
Nucleic acids
Ressonància magnètica nuclear
Resonancia magnètica nuclear (Física)
Nuclear magnetic resonance
Ciències Experimentals i Matemàtiques
547
id ES_091674aaa4003938ef338bc8a6cebf5b
oai_identifier_str oai:www.tdx.cat:10803/668126
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
title Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
spellingShingle Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
Mir Morro, Bartomeu
Química orgànica
Química orgánica
Organic chemistry
Àcids nucleics
Ácidos nucleicos
Nucleic acids
Ressonància magnètica nuclear
Resonancia magnètica nuclear (Física)
Nuclear magnetic resonance
Ciències Experimentals i Matemàtiques
547
title_short Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
title_full Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
title_fullStr Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
title_full_unstemmed Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
title_sort Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilization
dc.creator.none.fl_str_mv Mir Morro, Bartomeu
author Mir Morro, Bartomeu
author_facet Mir Morro, Bartomeu
author_role author
dc.contributor.none.fl_str_mv Escaja Sánchez, Nuria
Pedroso Muller, Enrique
Universitat de Barcelona. Departament de Química Inorgànica i Orgànica
dc.subject.none.fl_str_mv Química orgànica
Química orgánica
Organic chemistry
Àcids nucleics
Ácidos nucleicos
Nucleic acids
Ressonància magnètica nuclear
Resonancia magnètica nuclear (Física)
Nuclear magnetic resonance
Ciències Experimentals i Matemàtiques
547
topic Química orgànica
Química orgánica
Organic chemistry
Àcids nucleics
Ácidos nucleicos
Nucleic acids
Ressonància magnètica nuclear
Resonancia magnètica nuclear (Física)
Nuclear magnetic resonance
Ciències Experimentals i Matemàtiques
547
description Given the increasing interest in i-motif structures, obtaining such structures as well as detailed structural information under physiological conditions have become hot topics in the structural biology field. In this context, the main objectives of this thesis are focused on the design and detailed characterization of several oligonucleotide sequences that may form stable i-motif structures at neutral pH. The starting point is the mini i-motif structure, extensively studied in the research group that exhibits unusual high pH and thermal stability. These are dimeric structures stabilized by the formation of two hemiprotonated C:C+ base pairs capped at both ends by minor groove G:T:G:T tetrads. With the aim of getting deeper insights in this type of structures and enhance their stability at physiological conditions, different approaches were followed. A first strategy consisted in the incorporation of a neutral analogue of protonated cytidine (pseudoisocytidine, psC) occupying specific positions of the motif. The 3H-tautomer of psC, thanks to the extra hydrogen-bond donor, can form neutral base pairs completely isomorphic to hemiprotonated C:C+ pairs. psC was incorporated in different positions of dimeric mini i-motifs and in the telomeric sequence (HT0). The effect of the incorporation of psC depends on its position in the structure, being in most cases destabilizing. Neutral psC:C base pairs stabilize i-motifs at neutral pH, but the stabilization only occurs when psC:C base pairs are located at the ends of intercalated C:C+ stacks. Structural and stability data on the incorporation of pseudocytidine in i-motifs suggest that positively charged base pairs in the core of the structure are necessary to stabilize this non-canonical DNA structure. A second approach focused on exploring the compatibility of i-motif structures with other reported minor groove tetrads. The effect of different minor groove tetrads in i-motifs was studied in the context of short linear and cyclic oligonucleotides, affording dimeric mini i-motifs, but also in longer sequences that may form monomeric mini i-motif structures. The results show that the mini i-motif is compatible with different type of minor groove tetrads and a stability ranking could be established: G:C:G:T ≥ G:C:G:C >> G:T:G:T, exhibiting monomeric structures enhanced stability. Interestingly, a consensus sequence was outlined based on the results obtained for the set of mini i-motif-forming sequences. The mapping of this sequence throughout the human genome by bioinformatics analysis revealed a statistical prevalence. The distribution found for these sequences is not random, being much more frequent in regulatory regions Finally, a fluorescent cytidine analogue (1,3-diaza-2-oxophenoxazine, tCo), capable to hybridize as a cytosine and maintaining the ability to form Watson-Crick as well as hemiprotonated base pairs, was tested as an internal probe for the characterization of local environments within i-motif structures. NMR spectroscopy indicated both types of hybridization (tCo:C+ and G:tCo) are compatible with the mini i-motif structure at neutral pH. Interestingly, the fluorescence signal of tCo suffers a sever quenching when forming an hemiprotonated base pair, compared to the very little quenching that shows upon WC hybridization. Hence, tCo was successfully used as fluorescent probe for monitoring conformational transitions between different species of tCo-containing sequences. Moreover, when replacing a cytosine residue involved in a C:C+ pair, the favorable stacking between tCo and G:C base pairs from the capping minor groove assemblies provokes enhanced stability of the structure against both pH and temperature. Very remarkably, the visualization of mini i-motif structures in cellular media was accomplished by confocal fluorescence microscopy after the successful transfection of HeLa cells with tCo-containing oligonucleotides.
publishDate 2019
dc.date.none.fl_str_mv 2019
2019
2020
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
info:eu-repo/semantics/publishedVersion
format doctoralThesis
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10803/668126
url http://hdl.handle.net/10803/668126
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 326 p.
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Universitat de Barcelona
publisher.none.fl_str_mv Universitat de Barcelona
dc.source.none.fl_str_mv TDX (Tesis Doctorals en Xarxa)
reponame:TDR. Tesis Doctorales en Red
instname:CBUC, CESCA
instname_str CBUC, CESCA
reponame_str TDR. Tesis Doctorales en Red
collection TDR. Tesis Doctorales en Red
repository.name.fl_str_mv
repository.mail.fl_str_mv
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spelling Studies on the formation of i-motif structures at neutral pH. Use of cytidine analogues and importance of minor groove tetrads on mini i-motifs stabilizationMir Morro, BartomeuQuímica orgànicaQuímica orgánicaOrganic chemistryÀcids nucleicsÁcidos nucleicosNucleic acidsRessonància magnètica nuclearResonancia magnètica nuclear (Física)Nuclear magnetic resonanceCiències Experimentals i Matemàtiques547Given the increasing interest in i-motif structures, obtaining such structures as well as detailed structural information under physiological conditions have become hot topics in the structural biology field. In this context, the main objectives of this thesis are focused on the design and detailed characterization of several oligonucleotide sequences that may form stable i-motif structures at neutral pH. The starting point is the mini i-motif structure, extensively studied in the research group that exhibits unusual high pH and thermal stability. These are dimeric structures stabilized by the formation of two hemiprotonated C:C+ base pairs capped at both ends by minor groove G:T:G:T tetrads. With the aim of getting deeper insights in this type of structures and enhance their stability at physiological conditions, different approaches were followed. A first strategy consisted in the incorporation of a neutral analogue of protonated cytidine (pseudoisocytidine, psC) occupying specific positions of the motif. The 3H-tautomer of psC, thanks to the extra hydrogen-bond donor, can form neutral base pairs completely isomorphic to hemiprotonated C:C+ pairs. psC was incorporated in different positions of dimeric mini i-motifs and in the telomeric sequence (HT0). The effect of the incorporation of psC depends on its position in the structure, being in most cases destabilizing. Neutral psC:C base pairs stabilize i-motifs at neutral pH, but the stabilization only occurs when psC:C base pairs are located at the ends of intercalated C:C+ stacks. Structural and stability data on the incorporation of pseudocytidine in i-motifs suggest that positively charged base pairs in the core of the structure are necessary to stabilize this non-canonical DNA structure. A second approach focused on exploring the compatibility of i-motif structures with other reported minor groove tetrads. The effect of different minor groove tetrads in i-motifs was studied in the context of short linear and cyclic oligonucleotides, affording dimeric mini i-motifs, but also in longer sequences that may form monomeric mini i-motif structures. The results show that the mini i-motif is compatible with different type of minor groove tetrads and a stability ranking could be established: G:C:G:T ≥ G:C:G:C >> G:T:G:T, exhibiting monomeric structures enhanced stability. Interestingly, a consensus sequence was outlined based on the results obtained for the set of mini i-motif-forming sequences. The mapping of this sequence throughout the human genome by bioinformatics analysis revealed a statistical prevalence. The distribution found for these sequences is not random, being much more frequent in regulatory regions Finally, a fluorescent cytidine analogue (1,3-diaza-2-oxophenoxazine, tCo), capable to hybridize as a cytosine and maintaining the ability to form Watson-Crick as well as hemiprotonated base pairs, was tested as an internal probe for the characterization of local environments within i-motif structures. NMR spectroscopy indicated both types of hybridization (tCo:C+ and G:tCo) are compatible with the mini i-motif structure at neutral pH. Interestingly, the fluorescence signal of tCo suffers a sever quenching when forming an hemiprotonated base pair, compared to the very little quenching that shows upon WC hybridization. Hence, tCo was successfully used as fluorescent probe for monitoring conformational transitions between different species of tCo-containing sequences. Moreover, when replacing a cytosine residue involved in a C:C+ pair, the favorable stacking between tCo and G:C base pairs from the capping minor groove assemblies provokes enhanced stability of the structure against both pH and temperature. Very remarkably, the visualization of mini i-motif structures in cellular media was accomplished by confocal fluorescence microscopy after the successful transfection of HeLa cells with tCo-containing oligonucleotides.En el marc de les estructures no canòniques dels àcids nucleics i les seves possibles implicacions biològiques, aquesta tesi te com a objectiu principal l’obtenció i caracterització estructural d’estructures i- motif que siguin estables a pH neutre. El punt de partida és l’estructura mini i-motif, un tipus de motiu que presenta una inusual elevada estabilitat front pH i temperatura. Es tracta d’una estructura dimèrica estabilitzada per la formació de dos parells hemiprotonats C:C+ que interaccionen per apilament amb dues tètrades G:T:G:T de solc menor que actuen de tapa de l’estructura. Per a aprofundir en l’estudi d’aquest tipus de motius i augmentar-ne la seva estabilitat a pH fisiològic, diferents aproximacions s’han dut a terme. En la primera aproximació, s’ha explorat la incorporació d’un anàleg neutre de citidina protonada (pseudoisocitidina, psC), capaç de formar parells neutres isomòrfics als parells hemiprotonats C:C+. Els estudis realitzats demostren que l’efecte de la psC en depèn fortament de la posició dins l’estructura. La formació de parells hemiprotonats al centre de l’estructura és fonamental i la formació de parells neutres només és tolerada pels parells terminals, produint-se en aquest cas una certa estabilització. Una segona línia de treball s’ha centrat en explorar la compatibilitat d’estructures i-motif amb altres tètrades de solc menor. S’han estudiat diferents seqüències, lineals i cícliques, que donarien lloc a estructures mini i-motif amb diferents associacions de nucleobases a les tètrades. Els estudis demostren que el mini i-motif és compatible amb diferents tètrades de solc menor i s’ha pogut establir el següent rànquing d’estabilitat G:C:G:T ≥ G:C:G:C >> G:T:G:T. Cal destacar que, la seqüència consens per aquest tipus d’estructures s’ha determinat que és prevalent al genoma humà. Finalment, s’ha incorporat un anàleg de citidina fluorescent (1,3-diaza-2-oxofenoxazina, tCo), substituint citosines en posicions especifiques de l’estructura. Els resultats demostren que tCo actua de forma eficient com a sonda local per a monitoritzar transicions conformacionals i que, en posicions específiques te un efecte força positiu en l’estabilitat tèrmica del motiu. Aquest fet ha permès utilitzat un d’aquests derivats per a visualitzar el plegament de l’estructura en medi cel·lular.Universitat de BarcelonaEscaja Sánchez, NuriaPedroso Muller, EnriqueUniversitat de Barcelona. Departament de Química Inorgànica i Orgànica201920202019info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersion326 p.application/pdfapplication/pdfhttp://hdl.handle.net/10803/668126TDX (Tesis Doctorals en Xarxa)reponame:TDR. Tesis Doctorales en Redinstname:CBUC, CESCAInglésL'accés als continguts d'aquesta tesi queda condicionat a l'acceptació de les condicions d'ús establertes per la següent llicència Creative Commons: http://creativecommons.org/licenses/by-nc-nd/4.0/http://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:www.tdx.cat:10803/6681262026-06-14T12:46:07Z
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