Umeå University's logo

umu.sePublications
Change search
Link to record
Permanent link

Direct link
Publications (10 of 13) Show all publications
Sengupta, P., Jamroskovic, J. & Sabouri, N. (2023). A beginner's handbook to identify and characterize i-motif DNA. In: Hans Renata (Ed.), Methods in enzymology: . Elsevier
Open this publication in new window or tab >>A beginner's handbook to identify and characterize i-motif DNA
2023 (English)In: Methods in enzymology / [ed] Hans Renata, Elsevier, 2023Chapter in book (Refereed)
Abstract [en]

Genomic DNA exhibits an innate ability to manifest diverse sequence-dependent secondary structures, serving crucial functions in gene regulation and cellular equilibrium. While extensive research has confirmed the formation of G-quadruplex structures by guanine-rich sequences in vitro and in cells, recent investigations have turned the quadruplex community's attention to the cytosine (C)-rich complementary strands that can adopt unique tetra-stranded conformation, termed as intercalated motif or i-motif. I-motifs are stabilized by hemi-protonated C:CH+ base pairs under acidic conditions. Initially, the in vivo occurrence of i-motifs was underestimated because their formation is favored at non-physiological pH. However, groundbreaking research utilizing the structure-specific iMab antibody and high-throughput sequencing have recently detected their conserved dispersion throughout the genome, challenging previous assumptions. Given the evolving nature of this research field, it becomes imperative to conduct independent in vitro experiments aimed at identifying potential i-motif formation in C-rich sequences and consolidating the findings to address the properties of i-motifs. This chapter serves as an introductory guide for the swift identification of novel i-motifs, where we present an experimental framework for investigating and characterizing i-motif sequences in vitro. In this chapter, we selected a synthetic oligonucleotide (C7T3) sequence and outlined appropriate methodologies for annealing the i-motif structure into suitable buffers. Then, we validated its formation by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopy. Finally, we provided a thorough account of the step-by-step procedures to investigate the effect of i-motif formation on the stalling or retardation of DNA replication using high resolution primer extension assays.

Place, publisher, year, edition, pages
Elsevier, 2023
Series
Methods in enzymology, ISSN 0076-6879 ; 693
Keywords
Circular dichroism, I-motifs, Nuclear magnetic resonance, Primer extension assay, Quadruplex DNA
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-218112 (URN)10.1016/bs.mie.2023.11.001 (DOI)2-s2.0-85178602961 (Scopus ID)
Funder
Swedish Cancer Society, 22 2380 Pj 01HSwedish Research Council, VR-MH 2021-02468Knut and Alice Wallenberg Foundation, KAW 2021.0173The Kempe Foundations, SMK2058Wenner-Gren Foundations, o.UPD2020-0097Åke Wiberg Foundation, M20-0125
Available from: 2023-12-19 Created: 2023-12-19 Last updated: 2023-12-19
Jamroskovic, J., Deiana, M. & Sabouri, N. (2022). Probing the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution. Biochimie, 199, 81-91
Open this publication in new window or tab >>Probing the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution
2022 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 199, p. 81-91Article in journal (Refereed) Published
Abstract [en]

Cytosine-rich DNA can fold into four-stranded intercalated structures called i-motifs (iMs) under acidic conditions through the formation of hemi-protonated C:C+ base pairs. However, the folding and stability of iMs rely on many other factors that are not yet fully understood. Here, we combined biochemical and biophysical approaches to determine the factors influencing iM stability under a wide range of experimental conditions. By using high-resolution primer extension assays, circular dichroism, and absorption spectroscopies, we demonstrate that the stabilities of three different biologically relevant iMs are not dependent on molecular crowding agents. Instead, some of the crowding agents affected overall DNA synthesis. We also tested a range of small molecules to determine their effect on iM stabilization at physiological temperature and demonstrated that the G-quadruplex-specific molecule CX-5461 is also a promising candidate for selective iM stabilization. This work provides important insights into the requirements needed for different assays to accurately study iM stabilization, which will serve as important tools for understanding the contribution of iMs in cell regulation and their potential as therapeutic targets.

Place, publisher, year, edition, pages
Elsevier, 2022
Keywords
CX-5461, DNA replication, G-quadruplex DNA, High-resolution primer extension assay, I-motif DNA, pH
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-194635 (URN)10.1016/j.biochi.2022.04.007 (DOI)000800388200003 ()35452743 (PubMedID)2-s2.0-85129038456 (Scopus ID)
Funder
Swedish Research Council, 2021-02468Swedish Cancer Society, 2019/126Åke Wiberg Foundation, M20-0125Swedish Cancer Society, 21 0302 PT 01 H
Available from: 2022-05-12 Created: 2022-05-12 Last updated: 2023-09-05Bibliographically approved
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
Open this publication in new window or tab >>A Light‐up Logic Platform for Selective Recognition of Parallel G‐Quadruplex Structures via Disaggregation‐Induced Emission
Show others...
2020 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 59, no 2, p. 896-902Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2020
Keywords
aggregation, biosensor, DNA, G-quadruplex, logic gate
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-164662 (URN)10.1002/anie.201912027 (DOI)000497789300001 ()31644837 (PubMedID)2-s2.0-85075533982 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, KAW2015-0189Swedish Research Council, VR-NT 2017-05235Swedish Research Council, VR-MH 2018-02651The Kempe Foundations, SMK-1632
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2023-09-05Bibliographically approved
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
Open this publication in new window or tab >>A Site-Specific Self-Assembled Light-up Rotor Probe for Selective Recognition and Stabilization of c-MYC G-Quadruplex DNA
Show others...
2020 (English)In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 12, no 24, p. 12950-12957Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2020
National Category
Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Organic Chemistry Physical Chemistry
Research subject
Biochemistry; Organic Chemistry; Physical Chemistry; cell research
Identifiers
urn:nbn:se:umu:diva-171513 (URN)10.1039/D0NR03404E (DOI)000545599900025 ()2-s2.0-85087110627 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, KAW2015-0189Swedish Research Council, VR-NT 2017-05235Swedish Research Council, VR-MH 2018-02651The Kempe Foundations, SMK-1632
Available from: 2020-06-03 Created: 2020-06-03 Last updated: 2023-03-24Bibliographically approved
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
Open this publication in new window or tab >>Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization
Show others...
2020 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, no 6, p. 2876-2888Article in journal (Refereed) 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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Atom and Molecular Physics and Optics
Identifiers
urn:nbn:se:umu:diva-169314 (URN)10.1021/jacs.9b11232 (DOI)000514255300025 ()31990532 (PubMedID)2-s2.0-85079045732 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilThe Kempe Foundations, SMK-1632Åke Wiberg FoundationSwedish Cancer SocietyVästerbotten County Council, VLL-643451Västerbotten County Council, VLL-832001EU, Horizon 2020, 751474
Available from: 2020-03-31 Created: 2020-03-31 Last updated: 2023-03-24Bibliographically approved
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
Open this publication in new window or tab >>Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication
Show others...
2020 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, no 19, p. 10998-11015Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
Oxford University Press, 2020
National Category
Medical and Health Sciences
Research subject
molecular medicine (genetics and pathology); Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-176331 (URN)10.1093/nar/gkaa820 (DOI)000606018400033 ()2-s2.0-85095799661 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, KAW2015–0189Swedish Research Council, 2018–02651Swedish Research Council, 2017–05235Swedish Cancer Society, 2019/126Swedish Cancer Society, 2017/654The Kempe Foundations, SMK-1632Swedish Childhood Cancer Foundation, MT2016–0004Swedish Childhood Cancer Foundation, PR2019–0037Swedish Research Council, 2018–02651
Available from: 2020-10-29 Created: 2020-10-29 Last updated: 2023-03-24Bibliographically approved
Prasad, B., Das, R. N., Jamroskovic, J., Kumar, R., Hedenström, M., Sabouri, N. & Chorell, E. (2020). The Relation Between Position and Chemical Composition of Bis-Indole Substituents Determines Their Interactions With G-Quadruplex DNA. Chemistry - A European Journal, 26(43), 9561-9572
Open this publication in new window or tab >>The Relation Between Position and Chemical Composition of Bis-Indole Substituents Determines Their Interactions With G-Quadruplex DNA
Show others...
2020 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 26, no 43, p. 9561-9572Article in journal (Refereed) Published
Abstract [en]

G‐quadruplex (G4) DNA structures are linked to fundamental biological processes and human diseases, which has triggered the development of compounds that affect these DNA structures. However, more knowledge is needed about how small molecules interact with G4 DNA structures. This study describes the development of a new class of bis‐indoles (3,3‐diindolyl‐methyl derivatives) and detailed studies of how they interact with G4 DNA using orthogonal assays, biophysical techniques, and computational studies. This revealed compounds that strongly bind and stabilize G4 DNA structures, and detailed binding interactions which e.g. show that charge variance can play a key role in G4 DNA binding. Furthermore, the structure‐activity relationships generated opened the possibilities to replace or introduce new substituents on the core structure, which is of key importance to optimize compound properties or introduce probes to further expand the possibilities of these compounds as tailored research tools to study G4 biology.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2020
National Category
Chemical Engineering Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-171213 (URN)10.1002/chem.202000579 (DOI)000544966200001 ()32187406 (PubMedID)2-s2.0-85087461969 (Scopus ID)
Funder
The Kempe Foundations, SMK-1632Swedish Research Council, VR-MH 2018-2651Swedish Research Council, 2017-05235Knut and Alice Wallenberg Foundation, KAW2015-0189Swedish Cancer Society, CAN2019/126
Available from: 2020-05-28 Created: 2020-05-28 Last updated: 2023-03-24Bibliographically approved
Deiana, M., Jamroskovic, J., Obi, I. & Sabouri, N. (2020). Unravelling the cellular emission fingerprint of the benchmark G-quadruplex-interactive compound Phen-DC3. Chemical Communications, 56(91), 14251-14254
Open this publication in new window or tab >>Unravelling the cellular emission fingerprint of the benchmark G-quadruplex-interactive compound Phen-DC3
2020 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 56, no 91, p. 14251-14254Article in journal (Refereed) Published
Abstract [en]

Phen-DC3 is among the most commonly used G-quadruplex (G4)-stabilizers in vitro and in cells. Here, we show that the G4-interactive binding interactions enable one to tune the optical properties of Phen-DC3 allowing the detection of G4 structures in cancer cells. This work opens up new directions for the use of Phen-DC3 as a selective G4 fluorescent reporter.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2020
National Category
Medical and Health Sciences Natural Sciences
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-176333 (URN)10.1039/d0cc05483f (DOI)000590124000022 ()33118567 (PubMedID)2-s2.0-85096357070 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, KAW2015-0189Swedish Research Council, VR-MH 2018-02651Swedish Cancer Society, CAN19 0126
Available from: 2020-10-29 Created: 2020-10-29 Last updated: 2023-03-23Bibliographically approved
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.
Open this publication in new window or tab >>Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay
Show others...
2019 (English)In: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856, Vol. 82, article id 102678Article in journal (Refereed) 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.

Keywords
G-quadruplex DNA, Schizosaccharomyces pombe, G4 stabilizer PhenDC3, Pif1 family helicase Pfh1, DNA replication, Quantitative PCR
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-162927 (URN)10.1016/j.dnarep.2019.102678 (DOI)000491627000009 ()31473486 (PubMedID)2-s2.0-85071382717 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, KAW2015-0189The Kempe Foundations, SMK1449 and SMK1632Swedish Society for Medical Research (SSMF)
Available from: 2019-09-02 Created: 2019-09-02 Last updated: 2019-11-20Bibliographically approved
Prasad, B., Jamroskovic, J., Bhowmik, S., Kumar, R., Romell, T., Sabouri, N. & Chorell, E. (2018). Flexible Versus Rigid G-Quadruplex DNA Ligands: Synthesis of Two Series of Bis-indole Derivatives and Comparison of Their Interactions with G-Quadruplex DNA. Chemistry - A European Journal, 24(31), 7926-7938
Open this publication in new window or tab >>Flexible Versus Rigid G-Quadruplex DNA Ligands: Synthesis of Two Series of Bis-indole Derivatives and Comparison of Their Interactions with G-Quadruplex DNA
Show others...
2018 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 24, no 31, p. 7926-7938Article in journal (Refereed) Published
Abstract [en]

Small molecules that target G-quadruplex (G4) DNA structures are not only valuable to study G4 biology but also for their potential as therapeutics. This work centers around how different design features of small molecules can affect the interactions with G4 DNA structures, exemplified by the development of synthetic methods to bis-indole scaffolds. Our synthesized series of bis-indole scaffolds are structurally very similar but differ greatly in the flexibility of their core structures. The flexibility of the molecules proved to be an advantage compared to locking the compounds in the presumed bioactive G4 conformation. The flexible derivatives demonstrated similar or even improved G4 binding and stabilization in several orthogonal assays even though their entropic penalty of binding is higher. In addition, molecular dynamics simulations with the c-MYC G4 structure showed that the flexible compounds adapt better to the surrounding. This was reflected by an increased number of both stacking and polar interactions with both the residues in the G4 DNA structure and the DNA residues just upstream of the G4 structure.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018
Keywords
DNA structures, G-quadruplexes, bis-indoles, drug design, nitrogen heterocycles
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-148052 (URN)10.1002/chem.201800078 (DOI)000434216600019 ()29603472 (PubMedID)2-s2.0-85048327004 (Scopus ID)
Available from: 2018-06-14 Created: 2018-06-14 Last updated: 2018-11-01Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6871-7663

Search in DiVA

Show all publications