Interactions between a dsDNA Oligonucleotide and Imidazolium Chloride Ionic Liquids: Effect of Alkyl Chain Length, Part I

Ionic liquids (ILs) have become nearly ubiquitous solvents and their interactions with biomolecules has been a focus of study. Here, we used the fluorescence emission of DAPI, a groove binding fluorophore, coupled with molecular dynamics (MD) simulations to report on interactions between imidazolium chloride ([Imn,1]+) ionic liquids and a synthetic DNA oligonucleotide composed entirely of T×A bases (7(TA)) to elucidate the effects ILs on a model DNA duplex. Spectral shifts on the order of 500–1000 cm−1, spectral broadening (~1000 cm−1), and excitation and emission intensity ratio changes combine to give evidence of an increased DAPI environment heterogeneity on added IL. Fluorescence lifetimes for DAPI/IL solutions yielded two time constants 0.15 ns (~80% to 60% contribution) and 2.36–2.71 ns for IL up to 250 mM. With DNA, three time constants were required that varied with added IL (0.33–0.15 ns (1–58% contribution), ~1.7–1.0 ns (~5% contribution), and 3.8–3.6 ns (94–39% contribution)). MD radial distribution functions revealed that π-π stacking interactions between the imidazolium ring were dominant at lower IL concentration and that electrostatic and hydrophobic interactions become more prominent as IL concentration increased. Alkyl chain alignment with DNA and IL-IL interactions also varied with IL. Collectively, our data showed that, at low IL concentration, IL was primarily bound to the DNA minor groove and with increased IL concentration the phosphate regions and major groove binding sites were also important contributors to the complete set of IL-DNA duplex interactions.

Dissociation pathways of the p53 DNA binding domain from DNA and critical roles of key residues elucidated by dPaCS-MD/MSM

The dissociation process of the DNA binding domain of p53 (p53-DBD) from a DNA duplex that contains the consensus sequence, which is the specific target of p53-DBD, was investigated by a combination of dissociation parallel cascade selection molecular dynamics (dPaCS-MD) and the Markov state model (MSM). Based on an all-atom model including explicit solvent, we first simulated the p53-DBD dissociation processes by 75 trials of dPaCS-MD, which required an average simulation time of 11.2 ± 2.2 ns per trial. By setting the axis of the DNA duplex as the Z-axis and the binding side of p53-DBD on DNA as the + side of the X-axis, we found that dissociations took place along the +X and −Y directions (−Y directions) in 93% of the cases, while 7% of the cases moved along +X and +Y directions (+Y directions). Toward the −Y directions, p53-DBD dissociated first from the major groove and then detached from the minor groove, while unbinding from the minor groove occurred first in dissociations along the +Y directions. Analysis of the free energy landscape by MSM showed that loss of the minor groove interaction with p53-DBD toward the +Y directions incurred a relatively high energy cost (1.1 kcal/mol) upon a critical transition, whereas major groove detachment more frequently occurred with lower free energy costs. The standard binding free energy calculated from the free energy landscape was −10.9 ± 0.4 kcal/mol, which agrees with an experimental value of –11.1 kcal/mol. These results indicate that the dPaCS-MD/MSM combination can be a powerful tool to investigate dissociation mechanisms of two large molecules. Minor groove binding is mainly stabilized by R248, identified as the most important residue that tightly binds deep inside the minor groove. Analysis of the p53 key residues for DNA binding indicates high correlations with cancer-related mutations, confirming that impairment of the interactions between p53-DBD and DNA can be frequently related to cancer.

DNA Binding Mode Analysis of a Core-Extended Naphthalene Diimide as a Conformation-Sensitive Fluorescent Probe of G-Quadruplex Structures

G-quadruplex existence was proved in cells by using both antibodies and small molecule fluorescent probes. However, the G-quadruplex probes designed thus far are structure- but not conformation-specific. Recently, a core-extended naphthalene diimide (cex-NDI) was designed and found to provide fluorescent signals of markedly different intensities when bound to G-quadruplexes of different conformations or duplexes. Aiming at evaluating how the fluorescence behaviour of this compound is associated with specific binding modes to the different DNA targets, cex-NDI was here studied in its interaction with hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex models by biophysical techniques, molecular docking, and biological assays. cex-NDI showed different binding modes associated with different amounts of stacking interactions with the three DNA targets. The preferential binding sites were the groove, outer quartet, or intercalative site of the hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex, respectively. Interestingly, our data show that the fluorescence intensity of DNA-bound cex-NDI correlates with the amount of stacking interactions formed by the ligand with each DNA target, thus providing the rationale behind the conformation-sensitive properties of cex-NDI and supporting its use as a fluorescent probe of G-quadruplex structures. Notably, biological assays proved that cex-NDI mainly localizes in the G-quadruplex-rich nuclei of cancer cells.

Two coexisting pseudo-mirror heteromolecular telomeric G-quadruplexes in opposite loop progressions differentially recognized by a low equivalent of Thioflavin T

The final 3′-terminal residue of the telomeric DNA G-overhang is inherently less precise. Here, we describe how alteration of the last 3′-terminal base affects the mutual recognition between two different G-rich oligomers of human telomeric DNA in the formation of heteromolecular G-quadruplexes (hetero-GQs). Associations between three- and single-repeat fragments of human telomeric DNA, target d(GGGTTAGGGTTAGGG) and probe d(TAGGGT), in Na+ solution yield two coexisting forms of (3 + 1) hybrid hetero-GQs: the kinetically favourable LLP-form (left loop progression) and the thermodynamically controlled RLP-form (right loop progression). However, only the adoption of a single LLP-form has been previously reported between the same probe d(TAGGGT) and a target variant d(GGGTTAGGGTTAGGGT) having one extra 3′-end thymine. Moreover, the flanking base alterations of short G-rich probe variants also significantly affect the loop progressions of hetero-GQs. Although seemingly two pseudo-mirror counter partners, the RLP-form exhibits a preference over the LLP-form to be recognized by a low equivalent of fluorescence dye thioflavin T (ThT). To a greater extent, ThT preferentially binds to RLP hetero-GQ than with the corresponding telomeric DNA duplex context or several other representative unimolecular GQs.

Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.