In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells.

I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.

The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.

Understanding intercalative modulation of G-rich sequence folding: solution structure of a TINA-conjugated antiparallel DNA triplex.

We present here the high-resolution structure of an antiparallel DNA triplex in which a monomer of para-twisted intercalating nucleic acid (para-TINA: (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol) is covalently inserted as a bulge in the third strand of the triplex. TINA is a potent modulator of the hybridization properties of DNA sequences with extremely useful properties when conjugated in G-rich oligonucleotides. The insertion of para-TINA between two guanines of the triplex imparts a high thermal stabilization (ΔTM = 9ºC) to the structure and enhances the quality of NMR spectra by increasing the chemical shift dispersion of proton signals near the TINA location. The structural determination reveals that TINA intercalates between two consecutive triads, causing only local distortions in the structure. The two aromatic moieties of TINA are nearly coplanar, with the phenyl ring intercalating between the flanking guanine bases in the sequence, and the pyrene moiety situated between the Watson-Crick base pair of the two first strands. The precise position of TINA within the triplex structure reveals key TINA-DNA interactions, which explains the high stabilization observed and will aid in the design of new and more efficient binders to DNA.

pH-Dependent Capping Interactions Induce Large-Scale Structural Transitions in i-Motifs.

We study here a DNA oligonucleotide having the ability to form two different i-motif structures whose relative stability depends on pH and temperature. The major species at neutral pH is stabilized by two C:C+ base pairs capped by two minor groove G:C:G:C tetrads. The high pH and thermal stability of this structure are mainly due to the favorable effect of the minor groove tetrads on their adjacent positively charged C:C+ base pairs. At pH 5, we observe a more elongated i-motif structure consisting of four C:C+ base pairs capped by two G:T:G:T tetrads. Molecular dynamics calculations show that the conformational transition between the two structures is driven by the protonation state of key cytosines. In spite of large conformational differences, the transition between the acidic and neutral structures can occur without unfolding of the i-motif. These results represent the first case of a conformational switch between two different i-motif structures and illustrate the dramatic pH-dependent plasticity of this fascinating DNA motif.

Structure of i-Motif/Duplex Junctions at Neutral pH.

We report here the three-dimensional structure of an i-motif/duplex junction, determined by NMR methods at neutral pH. By including a minor groove tetrad at one side of the C:C+ stack of a monomeric i-motif, and a stem/loop hairpin at the other side, we have designed stable DNA constructs in which i-DNA and B-DNA regions coexist in a wide range of experimental conditions. This study demonstrates that i- and B-DNA are structurally compatible, giving rise to a distinctive fold with peculiar groove shapes. The effect of different residues at the i-motif/duplex interface has been explored. We also show that these constructs can be adapted to sequences of biological relevance, like that found in the promoter region of the KRAS oncogene.

Potential G-quadruplexes and i-Motifs in the SARS-CoV-2.

Quadruplex structures have been identified in a plethora of organisms where they play important functions in the regulation of molecular processes, and hence have been proposed as therapeutic targets for many diseases. In this paper we report the extensive bioinformatic analysis of the SARS-CoV-2 genome and related viruses using an upgraded version of the open-source algorithm G4-iM Grinder. This version improves the functionality of the software, including an easy way to determine the potential biological features affected by the candidates found. The quadruplex definitions of the algorithm were optimized for SARS-CoV-2. Using a lax quadruplex definition ruleset, which accepts amongst other parameters two residue G- and C-tracks, 512 potential quadruplex candidates were discovered. These sequences were evaluated by their in vitro formation probability, their position in the viral RNA, their uniqueness and their conservation rates (calculated in over seventeen thousand different COVID-19 clinical cases and sequenced at different times and locations during the ongoing pandemic). These results were then compared subsequently to other Coronaviridae members, other Group IV (+)ssRNA viruses and the entire viral realm. Sequences found in common with other viral species were further analyzed and characterized. Sequences with high scores unique to the SARS-CoV-2 were studied to investigate the variations amongst similar species. Quadruplex formation of the best candidates were then confirmed experimentally. Using NMR and CD spectroscopy, we found several highly stable RNA quadruplexes that may be suitable therapeutic targets for the SARS-CoV-2.

De Novo Design of Selective Quadruplex-Duplex Junction Ligands and Structural Characterisation of Their Binding Mode: Targeting the G4 Hot-Spot.

Invited for the cover of this issue are Andrés G. Santana, Carlos González, Juan Luis Asensio and co-workers at Instituto de Química Orgánica General, Instituto de Química-Física Rocasolano and Universidad de La Rioja. The image depicts drug selectivity using a metaphor of an arrow hitting a target. Read the full text of the article at 10.1002/chem.202005026.

De Novo Design of Selective Quadruplex-Duplex Junction Ligands and Structural Characterisation of Their Binding Mode: Targeting the G4 Hot-Spot.

Targeting the interface between DNA quadruplex and duplex regions by small molecules holds significant promise in both therapeutics and nanotechnology. Herein, a new pharmacophore is reported, which selectively binds with high affinity to quadruplex-duplex junctions, while presenting a poorer affinity for G-quadruplex or duplex DNA alone. Ligands complying with the reported pharmacophore exhibit a significant affinity and selectivity for quadruplex-duplex junctions, including the one observed in the HIV-1 LTR-III sequence. The structure of the complex between a quadruplex-duplex junction with a ligand of this family has been determined by NMR methods. According to these data, the remarkable selectivity of this structural motif for quadruplex-duplex junctions is achieved through an unprecedented interaction mode so far unexploited in medicinal and biological chemistry: the insertion of a benzylic ammonium moiety into the centre of the partially exposed G-tetrad at the interface with the duplex. Further decoration of the described scaffolds with additional fragments opens up the road to the development of selective ligands for G-quadruplex-forming regions of the genome.