Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino ¹H CEST

In duplex DNA, Watson–Crick A–T and G–C base pairs (bp's) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for identifying potential Hoogsteen hot spots and for understanding the potential roles of Hoogsteen base pairs in DNA recognition and repair. However, such studies are hampered by the need to prepare 13C or 15N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) 1H CEST experiments employing high-power radiofrequency fields (B1 > 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Watson–Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino 1H CEST were in very good agreement with counterparts measured using off-resonance 13C / 15N spin relaxation in the rotating frame (R1ρ). It is shown that 1H–1H NOE effects which typically introduce artifacts in 1H-based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short (≤ 100 ms). The 1H CEST experiment can be performed with ∼ 10× higher throughput and ∼ 100× lower cost relative to 13C / 15N R1ρ and enabled Hoogsteen chemical exchange measurements undetectable by R1ρ. The results reveal an increased propensity to form Hoogsteen bp's near terminal ends and a diminished propensity within A-tract motifs. The 1H CEST experiment provides a basis for rapidly screening Hoogsteen breathing in duplex DNA, enabling identification of unusual motifs for more in-depth characterization.

Selectivity of a bromoacridine-containing fluorophore for triplex DNA

Fluorophore 1,8-naphthilamide was linked to 2-bromoacridine through an ethylenediamine spacer using a succinct synthetic route to give a bromoacridine-linked, bifunctional fluorophore conjugate for the detection of triplex DNA. Acridine is well known to intercalate into duplex DNA whereas introduction of a bulky bromine atom at position C2 redirects specificity for triplex over duplex DNA. In this work, photoelectron transfer assay was used to demonstrate that the synthesised 2-bromoacridine-linked fluorophore conjugate had good selectivity for the representative triplex DNA target sequence d(T*A.T)20 compared with double-stranded d(T.A)20, single-stranded dT20 or d(G/A)19 DNA sequences. Graphic abstract

New Insights on the Interaction of Phenanthroline Based Ligands and Metal Complexes and Polyoxometalates with Duplex DNA and G-Quadruplexes

This work provides new insights from our team regarding advances in targeting canonical and non-canonical nucleic acid structures. This modality of medical treatment is used as a form of molecular medicine specifically against the growth of cancer cells. Nevertheless, because of increasing concerns about bacterial antibiotic resistance, this medical strategy is also being explored in this field. Up to three strategies for the use of DNA as target have been studied in our research lines during the last few years: (1) the intercalation of phenanthroline derivatives with duplex DNA; (2) the interaction of metal complexes containing phenanthroline with G-quadruplexes; and (3) the activity of Mo polyoxometalates and other Mo-oxo species as artificial phosphoesterases to catalyze the hydrolysis of phosphoester bonds in DNA. We demonstrate some promising computational results concerning the favorable interaction of these small molecules with DNA that could correspond to cytotoxic effects against tumoral cells and microorganisms. Therefore, our results open the door for the pharmaceutical and medical applications of the compounds we propose.

Rapid measurement of Watson-Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino ¹H CEST

In duplex DNA, Watson-Crick A-T and G-C base pairs (bps) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for understanding the role of these non-canonical bps in DNA recognition and repair. However, such studies are hampered by the need to prepare 13C or 15N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) 1H CEST experiments employing high-power radiofrequency fields (B1 > 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Waston-Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino 1H CEST were in very good agreement with counterparts measured using off-resonance 13C/15N spin relaxation in the rotating frame (R1ρ). It is shown that 1H-1H NOE effects which typically introduce artifacts in 1H based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short (≤ 100 ms). The 1H CEST experiment can be performed with ~10X higher throughput and ~100X lower cost relative to 13C/15N R1ρ, and enabled Hoogsteen chemical exchange measurements undetectable by R1ρ. The results reveal an increased propensity to form Hoogsteen bps near terminal ends and a diminished propensity within A-tract motifs. The 1H CEST experiment opens the door to more comprehensively characterizing Hoogsteen breathing in duplex DNA.

Towards a Structural Mechanism for Sister Chromatid Cohesion Establishment at the Eukaryotic Replication Fork

Cohesion between replicated chromosomes is essential for chromatin dynamics and equal segregation of duplicated genetic material. In the G1 phase, the ring-shaped cohesin complex is loaded onto duplex DNA, enriching at replication start sites, or “origins”. During the same phase of the cell cycle, and also at the origin sites, two MCM helicases are loaded as symmetric double hexamers around duplex DNA. During the S phase, and through the action of replication factors, cohesin switches from encircling one parental duplex DNA to topologically enclosing the two duplicated DNA filaments, which are known as sister chromatids. Despite its vital importance, the structural mechanism leading to sister chromatid cohesion establishment at the replication fork is mostly elusive. Here we review the current understanding of the molecular interactions between the replication machinery and cohesin, which support sister chromatid cohesion establishment and cohesin function. In particular, we discuss how cryo-EM is shedding light on the mechanisms of DNA replication and cohesin loading processes. We further expound how frontier cryo-EM approaches, combined with biochemistry and single-molecule fluorescence assays, can lead to understanding the molecular basis of sister chromatid cohesion establishment at the replication fork.