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A beginner's handbook to identify and characterize i-motif DNA
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.ORCID iD: 0000-0001-6871-7663
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.ORCID iD: 0000-0002-4541-7702
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 [en]
Circular dichroism, I-motifs, Nuclear magnetic resonance, Primer extension assay, Quadruplex DNA
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-218112DOI: 10.1016/bs.mie.2023.11.001Scopus ID: 2-s2.0-85178602961OAI: oai:DiVA.org:umu-218112DiVA, id: diva2:1821110
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, M20-0125Available from: 2023-12-19 Created: 2023-12-19 Last updated: 2023-12-19

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Sengupta, PallabiJamroskovic, JanSabouri, Nasim

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Total: 199 hits
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