1. Academic Validation
  2. Probing the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution

Probing the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution

  • Biochimie. 2022 Aug;199:81-91. doi: 10.1016/j.biochi.2022.04.007.
Jan Jamroskovic 1 Marco Deiana 2 Nasim Sabouri 3
Affiliations

Affiliations

  • 1 Department of Medical Biochemistry and Biophysics, Umeå University, 901 87, Umeå, Sweden. Electronic address: jan.jamroskovic@umu.se.
  • 2 Department of Medical Biochemistry and Biophysics, Umeå University, 901 87, Umeå, Sweden.
  • 3 Department of Medical Biochemistry and Biophysics, Umeå University, 901 87, Umeå, Sweden. Electronic address: nasim.sabouri@umu.se.
Abstract

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.

Keywords

CX-5461; DNA replication; G-quadruplex DNA; High-resolution primer extension assay; I-motif DNA; pH.

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