Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells

Abstract

C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures in vivo has been missing. Whether i-motif structures form in complex environment of living cells is not currently known. Herein, using state-of-the-art in-cell NMR spectroscopy, we evaluate the stabilities of i-motif structures in the complex cellular environment. We show that i-motifs formed from naturally occurring C-rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i-motif stabilities in vivo are generally distinct from those in vitro. Our results are the first to interlink the stability of DNA i-motifs in vitro with their stability in vivo and provide essential information for the design and development of i-motif-based DNA biosensors for intracellular applications.

Keywords: DNA; i-motifs; in-cell NMR spectroscopy; structural biology.

Figure 1

A) Schematic of an intramolecular i‐motif structure and B) C.C⁺ base pair according to Lieblein et al.2c.

Figure 2

A) Double‐staining (PI/FAM) FCM analysis of transfected HeLa cells with the (FAM)‐DAP construct (upper left corner). Percentages of viable DNA non‐transfected cells, viable DNA‐containing cells, non‐transfected dead/compromised cells, and transfected dead/compromised cells with DNA are indicated in left‐bottom, right‐bottom, left‐top, and right‐top quadrants, respectively. Confocal microscope images of cells transfected with (FAM)‐DAP (upper right corner). The green color indicates the localization of (FAM)‐DAP. The blue color corresponds to a cell nucleus stained by Hoechst 33342. Imino region of 1D ¹H NMR spectra of DAP in vitro in T‐buffer (140 mm sodium phosphate, 5 mm KCl, 10 mm MgCl₂, pH 7.0) (black) and in‐cell (red). Imino region of 1D ¹H NMR spectrum of extracellular fluid (Leibovitz L15 medium) taken from the in‐cell NMR samples after completion of the spectra acquisition (gray). The (in‐cell) NMR spectra were acquired at 20 °C. B–D) Analogous data for HIF‐1α, PDGF‐A, and JAZF1. For the extended version of Figure 2 including all controls, see Figure S4.

Figure 3

Imino region of 1D ¹H NMR spectra acquired at various temperatures under in vitro conditions in T‐buffer (TB: 140 mm sodium phosphate, 5 mm KCl, 10 mm MgCl₂, pH 7.0) in green, in IC buffer (ICB: 25 mm potassium phosphate, 85 mm KCl, 10 mm NaCl, 1 mm MgCl₂,130 nm CaCl₂, pH 7.2) in black, and under in vivo conditions (red) for A) DAP, B) HIF‐1α, C) PDGF‐A, and D) JAZF1. Blue spectra were acquired at 20 °C, 30 min after the exposure of in vitro/in vivo samples to 40 °C. The PDGF‐A data are from two independent experiments. The data from second experiment are marked with asterisks. For controls relevant to temperature jump experiment see Figure S4.

Figure 4

Normalized molar ellipticities (left panel) extracted from the CD spectra for the A) DAP and B) JAZF1 i‐motif constructs recorded at signal maximum in the range between 285–290 nm as a function of temperature in IC buffer (25 mm potassium phosphate, 85 mm KCl, 10 mm NaCl, 1 mm MgCl₂,130 nm CaCl₂, pH 7.0) in the absence (green) and the presence of either 20 % glycerol (blue) or 20 % Ficoll 70 (black). Imino region of 1D ¹H NMR spectra (three panels on the right) recorded for the A) DAP and B) JAZF1 i‐motif constructs as a function of the temperature under identical conditions.