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.

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

“…[1] Such failures are often linked to poor membrane permeability and/ or to the lack of binding specificity in the cellular environment. Nuclear magnetic resonance (NMR) spectroscopy applied to living cells [2][3][4][5][6] has the potential to overcome these critical bottlenecks, as it can directly observe macromolecule-ligand interactions at atomic resolution within the cellular environment. [2,7,8] Herein, we report an approach to perform protein-observed ligand screening by NMR directly in the cytosol of living human cells.…”

Effects of Pressure and pH on the Physical Stability of an I‐Motif DNA Structure

“…[12b,13] Moreover, proteins that bind specifically to C-rich DNA sequences in vitro have been identified, [14] The stability of exogeneously provided imotifs in mammalian nuclei has recently been established and contrasted with in vitro stability. [15] Various in vivo functional roles for i-motifs have been proposed, [16] and recently the presence of endogenous i-motif in vivo has been confirmed, and a role in cell-cycle processes proposed. [17] While some research has been performed on the stability of i-motif structures as a limited function of pH and molecular crowding at ambient pressure, [11] there are currently only two publications of which we are aware that examine the effects of pressure on the thermal stability of an i-motif [10a,18] and these were limited to pHs well above the pK a of a [C..H..C] + pair.…”

Observing an antisense drug complex in intact human cells by in-cell NMR

“…Interestingly,t hese chemically tagged ASOs have significantly poorer downregulation properties than the regular untagged ASO, as shown by quantitative reverse transcription polymerase chain reaction( qRT-PCR) for pharmaceutically relevant doses of 1.25 mm [(67.9 AE 5.1) %f or unmodified ASO, (34.9 AE 8.9) %f or biotin-labelled and (37 AE 17) %f or Cy3-labelledA SO, Figure 2E;d etails in Sections 1.9, 1.10, 2.1-2.3, 2.6, 2.7 in the Supporting Information]. Because the tagged versions are significantly less efficient, they might not reflect how untagged ASO behaves in the cell, thus highlighting the problem that drug modifications with, for example, fluorescent or biotin tags can alter activity or localization and emphasizingt he need for at ag-free technique.In-cell NMR spectroscopy is at ag-free technique and allows the study,a ta tomic resolution,o fs tructural properties of, for example, proteins [7][8][9][10][11][12][13] and small DNA/RNA hairpins [14][15][16][17] in cells. Therefore, the ASO was delivered into HEK293T cells by electroporation,a su sed previously fors olution-state in-cell NMR studies, [9,17] to ensure robust uptake, and ac lassical in-cell solution-state NMR spectrum was acquired.…”

“…In addition to the 1D CP spectrum,a 2D 1 H, 31 PC P-PASS [29] in-cell spectrum could be acquired within 2h 40 min to verify the isotropic chemical shift of the bound ASO ( Figure 3C). Although solution-state in-cell NMR investigation of transfected oligonucleotides has been carried out before, [14][15][16][17] the ASO investigated hered id not produce any signal detectable by this technique.T his suggestst hat the amount of free intracellular ASO is low and that the ASO insteade xists in the form of macromolecular complexes with mRNA and/ord ifferent proteins, as might be expectedf rom the literature. [2,4,18] To overcome the current limitations of solution-state in-cell NMR (sensitivity,s tabilitya nd size) we presentafrozen in-cell DNP approach.T ot he best of our knowledge,t his is the first time that DNP has been used to detect an exogenous oligonucleotide delivered into cells, and also the first time that at ransfected antisense drug in macromolecular complexes has been detected in intact human cells.…”

“…In-cell NMR spectroscopy is at ag-free technique and allows the study,a ta tomic resolution,o fs tructural properties of, for example, proteins [7][8][9][10][11][12][13] and small DNA/RNA hairpins [14][15][16][17] in cells. Therefore, the ASO was delivered into HEK293T cells by electroporation,a su sed previously fors olution-state in-cell NMR studies, [9,17] to ensure robust uptake, and ac lassical in-cell solution-state NMR spectrum was acquired. However, in the 31 PNMR spectrumn oA SO signal could be detected (Figure 2A).…”