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Berner, A., Das, R. N., Bhuma, N., Golebiewska, J., Abrahamsson, A., Andréasson, M., . . . Chorell, E. (2023). G4-ligand-conjugated oligonucleotides mediate selective binding and stabilization of individual G4 DNA structures. Journal of the American Chemical Society, 146(10), 6926-6935
Öppna denna publikation i ny flik eller fönster >>G4-ligand-conjugated oligonucleotides mediate selective binding and stabilization of individual G4 DNA structures
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2023 (Engelska)Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 146, nr 10, s. 6926-6935Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2023
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
urn:nbn:se:umu:diva-222294 (URN)10.1021/jacs.3c14408 (DOI)001179314400001 ()38430200 (PubMedID)2-s2.0-85186374110 (Scopus ID)
Forskningsfinansiär
Kempestiftelserna, JCK-3159Kempestiftelserna, SMK-1632Kempestiftelserna, SMK21-0059Vetenskapsrådet, 2017-05235Vetenskapsrådet, 2021-04805Vetenskapsrådet, 2018-0278Cancerforskningsfonden i Norrland, AMP19-968Knut och Alice Wallenbergs Stiftelse, SMK21-0059
Tillgänglig från: 2024-03-20 Skapad: 2024-03-20 Senast uppdaterad: 2024-03-20Bibliografiskt granskad
Deiana, M., Chand, K., Chorell, E. & Sabouri, N. (2023). Parallel G-quadruplex DNA structures from nuclear and mitochondrial genomes trigger emission enhancement in a nonfluorescent nano-aggregated fluorine-boron-based dye. Journal of Physical Chemistry Letters, 14(7), 1862-1869
Öppna denna publikation i ny flik eller fönster >>Parallel G-quadruplex DNA structures from nuclear and mitochondrial genomes trigger emission enhancement in a nonfluorescent nano-aggregated fluorine-boron-based dye
2023 (Engelska)Ingår i: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 14, nr 7, s. 1862-1869Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Molecular self-assembly is a powerful tool for the development of functional nanostructures with adaptive optical properties. However, in aqueous solution, the hydrophobic effects in the monomeric units often afford supramolecular architectures with typical side-by-side π-stacking arrangement with compromised emissive properties. Here, we report on the role of parallel DNA guanine quadruplexes (G4s) as supramolecular disaggregating-capture systems capable of coordinating a zwitterionic fluorine-boron-based dye and promoting activation of its fluorescence signal. The dye's high binding affinity for parallel G4s compared to nonparallel topologies leads to a selective disassembly of the dye's supramolecular state upon contact with parallel G4s. This results in a strong and selective disaggregation-induced emission that signals the presence of parallel G4s observable by the naked eye and inside cells. The molecular recognition strategy reported here will be useful for a multitude of affinity-based applications with potential in sensing and imaging systems.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2023
Nationell ämneskategori
Fysikalisk kemi
Identifikatorer
urn:nbn:se:umu:diva-205385 (URN)10.1021/acs.jpclett.2c03301 (DOI)000936590300001 ()36779779 (PubMedID)2-s2.0-85148521026 (Scopus ID)
Tillgänglig från: 2023-03-10 Skapad: 2023-03-10 Senast uppdaterad: 2023-09-05Bibliografiskt granskad
Bhuma, N., Chand, K., Andréasson, M., Mason, J. E., Das, R. N., Patel, A. K., . . . Chorell, E. (2023). The effect of side chain variations on quinazoline-pyrimidine G-quadruplex DNA ligands. European Journal of Medicinal Chemistry, 248, Article ID 115103.
Öppna denna publikation i ny flik eller fönster >>The effect of side chain variations on quinazoline-pyrimidine G-quadruplex DNA ligands
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2023 (Engelska)Ingår i: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 248, artikel-id 115103Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

G-quadruplex (G4) DNA structures are involved in central biological processes such as DNA replication and transcription. These DNA structures are enriched in promotor regions of oncogenes and are thus promising as novel gene silencing therapeutic targets that can be used to regulate expression of oncoproteins and in particular those that has proven hard to drug with conventional strategies. G4 DNA structures in general have a well-defined and hydrophobic binding area that also is very flat and featureless and there are ample examples of G4 ligands but their further progression towards drug development is limited. In this study, we use synthetic organic chemistry to equip a drug-like and low molecular weight central fragment with different side chains and evaluate how this affect the compound's selectivity and ability to bind and stabilize G4 DNA. Furthermore, we study the binding interactions of the compounds and connect the experimental observations with the compound's structural conformations and electrostatic potentials to understand the basis for the observed improvements. Finally, we evaluate the top candidates' ability to selectively reduce cancer cell growth in a 3D co-culture model of pancreatic cancer which show that this is a powerful approach to generate highly active and selective low molecular weight G4 ligands with a promising therapeutic window.

Ort, förlag, år, upplaga, sidor
Elsevier, 2023
Nationell ämneskategori
Läkemedelskemi
Identifikatorer
urn:nbn:se:umu:diva-202112 (URN)10.1016/j.ejmech.2023.115103 (DOI)000922160800001 ()2-s2.0-85146280645 (Scopus ID)
Forskningsfinansiär
Kempestiftelserna, SMK-1632Vetenskapsrådet, 2017–05235Vetenskapsrådet, 2017- 01531Sveriges läkarförbund, SLS-890521Region Västerbotten, RV-930167Knut och Alice Wallenbergs StiftelseMarianne och Marcus Wallenbergs Stiftelse, 2020.0189Cancerfonden, 20 1339 PjFCancerforskningsfonden i Norrland, LP 21–2298Cancerforskningsfonden i Norrland, LP 22–2332
Anmärkning

Originally included in thesis in manuscript form.

Tillgänglig från: 2023-01-02 Skapad: 2023-01-02 Senast uppdaterad: 2023-09-05Bibliografiskt granskad
Deiana, M., Obi, I., Andréasson, M., Tamilselvi, S., Chand, K., Chorell, E. & Sabouri, N. (2021). A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures. ACS Chemical Biology, 16(8), 1365-1376
Öppna denna publikation i ny flik eller fönster >>A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures
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2021 (Engelska)Ingår i: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 16, nr 8, s. 1365-1376Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2021
Nationell ämneskategori
Läkemedelskemi Biokemi och molekylärbiologi Biofysik
Identifikatorer
urn:nbn:se:umu:diva-187118 (URN)10.1021/acschembio.1c00134 (DOI)000697396400009 ()34328300 (PubMedID)2-s2.0-85113337330 (Scopus ID)
Forskningsfinansiär
Kempestiftelserna, SMK-1632Knut och Alice Wallenbergs Stiftelse, KAW2015-0189Cancerfonden, CAN 2019/126Vetenskapsrådet, 2017-05235Vetenskapsrådet, 2018-02651
Tillgänglig från: 2021-09-01 Skapad: 2021-09-01 Senast uppdaterad: 2023-03-23Bibliografiskt granskad
Deiana, M., Chand, K., Jamroskovic, J., Obi, I., Chorell, E. & Sabouri, N. (2020). A Light‐up Logic Platform for Selective Recognition of Parallel G‐Quadruplex Structures via Disaggregation‐Induced Emission. Angewandte Chemie International Edition, 59(2), 896-902
Öppna denna publikation i ny flik eller fönster >>A Light‐up Logic Platform for Selective Recognition of Parallel G‐Quadruplex Structures via Disaggregation‐Induced Emission
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2020 (Engelska)Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 59, nr 2, s. 896-902Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The design of turn‐on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G‐quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition‐driven disaggregation (on‐signal) of an ultrabright coumarin‐quinazoline conjugate. The synthesized probe selectively lights‐up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label free molecular logic system. Finally, our molecule preferentially stains the G4‐rich nucleoli of cancer cells.

Ort, förlag, år, upplaga, sidor
Wiley-VCH Verlagsgesellschaft, 2020
Nyckelord
aggregation, biosensor, DNA, G-quadruplex, logic gate
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
medicinsk biokemi
Identifikatorer
urn:nbn:se:umu:diva-164662 (URN)10.1002/anie.201912027 (DOI)000497789300001 ()31644837 (PubMedID)2-s2.0-85075533982 (Scopus ID)
Forskningsfinansiär
Knut och Alice Wallenbergs Stiftelse, KAW2015-0189Vetenskapsrådet, VR-NT 2017-05235Vetenskapsrådet, VR-MH 2018-02651Kempestiftelserna, SMK-1632
Tillgänglig från: 2019-10-25 Skapad: 2019-10-25 Senast uppdaterad: 2023-09-05Bibliografiskt granskad
Deiana, M., Chand, K., Jamroskovic, J., Das, R. N., Obi, I., Chorell, E. & Sabouri, N. (2020). A Site-Specific Self-Assembled Light-up Rotor Probe for Selective Recognition and Stabilization of c-MYC G-Quadruplex DNA. Nanoscale, 12(24), 12950-12957
Öppna denna publikation i ny flik eller fönster >>A Site-Specific Self-Assembled Light-up Rotor Probe for Selective Recognition and Stabilization of c-MYC G-Quadruplex DNA
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2020 (Engelska)Ingår i: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 12, nr 24, s. 12950-12957Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Direct and unambiguous evidence of the formation of G-quadruplexes (G4s) in human cells have shown their implication in several key biological events and has emphasized their role as important targets for small-molecule cancer therapeutics. Here, we report on the first example of a self-assembled multitasking molecular-rotor G4-binder able to discriminate between an extensive panel of G4 and non-G4 structures and to selectively light-up (up to 105-fold), bind (nanomolar range), and stabilize the c-MYC promoter G4 DNA. In particular, association with the c-MYC G4 triggers the disassembly of its supramolecular state (disaggregation-induced emission, DIE) and induces geometrical restrictions (motion-induced change in emission, MICE) leading to a significant enhancement of its emission yield. Moreover, this optical reporter is able to selectively stabilize the c-MYC G4 and inhibit DNA synthesis. Finally, by using confocal laser-scanning microscopy (CLSM) we show the ability of this compound to localize primarily in the subnuclear G4-rich compartments of cancer cells. This work provides a benchmark for the future design and development of a new generation of smart sequence-selective supramolecular G4-binders that combine outstanding sensing and stability properties, to be utilized in anti-cancer therapy.

Ort, förlag, år, upplaga, sidor
Royal Society of Chemistry, 2020
Nationell ämneskategori
Biokemi och molekylärbiologi Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci) Organisk kemi Fysikalisk kemi
Forskningsämne
biokemi; organisk kemi; fysikalisk kemi; cellforskning
Identifikatorer
urn:nbn:se:umu:diva-171513 (URN)10.1039/D0NR03404E (DOI)000545599900025 ()2-s2.0-85087110627 (Scopus ID)
Forskningsfinansiär
Knut och Alice Wallenbergs Stiftelse, KAW2015-0189Vetenskapsrådet, VR-NT 2017-05235Vetenskapsrådet, VR-MH 2018-02651Kempestiftelserna, SMK-1632
Tillgänglig från: 2020-06-03 Skapad: 2020-06-03 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Jamroskovic, J., Doimo, M., Chand, K., Obi, I., Kumar, R., Brännström, K., . . . Sabouri, N. (2020). Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization. Journal of the American Chemical Society, 142(6), 2876-2888
Öppna denna publikation i ny flik eller fönster >>Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization
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2020 (Engelska)Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, nr 6, s. 2876-2888Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci) Atom- och molekylfysik och optik
Identifikatorer
urn:nbn:se:umu:diva-169314 (URN)10.1021/jacs.9b11232 (DOI)000514255300025 ()31990532 (PubMedID)2-s2.0-85079045732 (Scopus ID)
Forskningsfinansiär
Knut och Alice Wallenbergs StiftelseVetenskapsrådetKempestiftelserna, SMK-1632Åke Wibergs StiftelseCancerfondenVästerbottens läns landsting, VLL-643451Västerbottens läns landsting, VLL-832001EU, Horisont 2020, 751474
Tillgänglig från: 2020-03-31 Skapad: 2020-03-31 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Obi, I., Rentoft, M., Singh, V., Jamroskovic, J., Chand, K., Chorell, E., . . . Sabouri, N. (2020). Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication. Nucleic Acids Research, 48(19), 10998-11015
Öppna denna publikation i ny flik eller fönster >>Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication
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2020 (Engelska)Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, nr 19, s. 10998-11015Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

G-quadruplex (G4) structures are stable noncanonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancerassociated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.

Ort, förlag, år, upplaga, sidor
Oxford University Press, 2020
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
molekylär medicin (genetik och patologi); medicinsk biokemi
Identifikatorer
urn:nbn:se:umu:diva-176331 (URN)10.1093/nar/gkaa820 (DOI)000606018400033 ()2-s2.0-85095799661 (Scopus ID)
Forskningsfinansiär
Knut och Alice Wallenbergs Stiftelse, KAW2015–0189Vetenskapsrådet, 2018–02651Vetenskapsrådet, 2017–05235Cancerfonden, 2019/126Cancerfonden, 2017/654Kempestiftelserna, SMK-1632Barncancerfonden, MT2016–0004Barncancerfonden, PR2019–0037Vetenskapsrådet, 2018–02651
Tillgänglig från: 2020-10-29 Skapad: 2020-10-29 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Kumar, R., Chand, K., Bhowmik, S., Das, R. N., Bhattacharjee, S., Hedenström, M. & Chorell, E. (2020). Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes. Nucleic Acids Research, 48(3), 1108-1119
Öppna denna publikation i ny flik eller fönster >>Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes
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2020 (Engelska)Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, nr 3, s. 1108-1119Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

G-quadruplex (G4) DNA structures are linked to key biological processes and human diseases. Small molecules that target specific G4 DNA structures and signal their presence would therefore be of great value as chemical research tools with potential to further advance towards diagnostic and therapeutic developments. However, the development of these types of specific compounds remain as a great challenge. In here, we have developed a compound with ability to specifically signal a certain c-MYC G4 DNA structure through a fluorescence light-up mechanism. Despite the compound's two binding sites on the G4 DNA structure, only one of them result in the fluorescence light-up effect. This G-tetrad selectivity proved to originate from a difference in flexibility that affected the binding affinity and tilt the compound out of the planar conformation required for the fluorescence light-up mechanism. The intertwined relation between the presented factors is likely the reason for the lack of examples using rational design to develop compounds with turn-on emission that specifically target certain G4 DNA structures. However, this study shows that it is indeed possible to develop such compounds and present insights into the molecular details of specific G4 DNA recognition and signaling to advance future studies of G4 biology.

Ort, förlag, år, upplaga, sidor
Oxford University Press, 2020
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-168878 (URN)10.1093/nar/gkz1205 (DOI)000515121900015 ()31912160 (PubMedID)2-s2.0-85082145471 (Scopus ID)
Forskningsfinansiär
Vetenskapsrådet, VR-NT 2017-05235Kempestiftelserna, SMK-1632
Tillgänglig från: 2020-03-19 Skapad: 2020-03-19 Senast uppdaterad: 2023-03-23Bibliografiskt granskad
Jamroskovic, J., Obi, I., Movahedi, A., Chand, K., Chorell, E. & Sabouri, N. (2019). Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay. DNA Repair, 82, Article ID 102678.
Öppna denna publikation i ny flik eller fönster >>Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay
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2019 (Engelska)Ingår i: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 82, artikel-id 102678Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

In order to understand in which biological processes the four-stranded G-quadruplex (G4) DNA structures play a role, it is important to determine which predicted regions can actually adopt a G4 structure. Here, to identify DNA regions in Schizosaccharomyces pombe that fold into G4 structures, we first optimized a quantitative PCR (qPCR) assay using the G4 stabilizer, PhenDC3. We call this method the qPCR stop assay, and used it to screen for G4 structures in genomic DNA. The presence of G4 stabilizers inhibited DNA amplification in 14/15 unexplored genomic regions in S. pombe that encompassed predicted G4 structures, suggesting that at these sites the stabilized G4 structure formed an obstacle for the DNA polymerase. Furthermore, the formation of G4 structures was confirmed by complementary in vitro assays. In vivo, the S. pombe G4 unwinder Pif1 helicase, Pfh1, was associated with tested G4 sites, suggesting that the G4 structures also formed in vivo. Thus, we propose that the confirmed G4 structures in S. pombe form an obstacle for replication in vivo, and that the qPCR stop assay is a method that can be used to identify G4 structures. Finally, we suggest that the qPCR stop assay can also be used for identifying G4 structures in other organisms, as well as being adapted to screen for novel G4 stabilizers.

Nyckelord
G-quadruplex DNA, Schizosaccharomyces pombe, G4 stabilizer PhenDC3, Pif1 family helicase Pfh1, DNA replication, Quantitative PCR
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:umu:diva-162927 (URN)10.1016/j.dnarep.2019.102678 (DOI)000491627000009 ()31473486 (PubMedID)2-s2.0-85071382717 (Scopus ID)
Forskningsfinansiär
Knut och Alice Wallenbergs Stiftelse, KAW2015-0189Kempestiftelserna, SMK1449 and SMK1632Svenska Sällskapet för Medicinsk Forskning (SSMF)
Tillgänglig från: 2019-09-02 Skapad: 2019-09-02 Senast uppdaterad: 2019-11-20Bibliografiskt granskad
Organisationer
Identifikatorer
ORCID-id: ORCID iD iconorcid.org/0000-0001-7691-4392

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