QN-302 demonstrates opposing effects between i-motif and G-quadruplex DNA structures in the promoter of the S100P gene

GC-rich sequences can fold into G-quadruplexes and i-motifs and are known to control gene expression in many organisms. The potent G-quadruplex experimental anticancer drug QN-302 down-regulates a number of cancer-related genes, in particular S100P. Here we show this ligand has strong opposing effects with i-motif DNA structures and is one of the most potent i-motif destabilising agents reported to date. QN-302 down-regulates the expression of numerous cancer-related genes by pan-quadruplex targeting. QN-302 exhibits exceptional combined synergistic effects compared to many other G-quadruplex and i-motif interacting compounds. This work further emphasises the importance of considering G-quadruplex and i-motif DNA structures as one dynamic system

Identification of sugar-containing natural products that interact with i-motif DNA

There are thousands of compounds shown to interact with G-quadruplex DNA, yet very few which target i-motif (iM) DNA. Previous work showed that tobramycin can interact with iM- DNA, indicating the potential for sugar-molecules to target these structures. Computational approaches indicated that the sugar-containing natural products baicalin and geniposidic acid had potential to target iM-DNA. We assessed the DNA interacting properties of these compounds using FRET-based DNA melting and a fluorescence-based displacement assay using iM-DNA structures from the human telomere and the insulin linked polymorphic region (ILPR), as well as complementary G-quadruplex and double stranded DNA. Both baicalin and geniposidic acid show promise as iM-interacting compounds with potential for use in experiments into the structure and function of i-motif forming DNA sequences and present starting points for further synthetic development of these as probes for iM-DNA

Anticancer activity and DNA interaction of bis(pyridyl)allene-derived metal complexes

The constant need for novel drugs has prompted the scientific community to explore alternative structures to natural products and small and medium size organic compounds used in classic medicinal and pharmaceutical chemistry. Since the discovery of cisplatin, organometallic compounds have revealed great potential as metallodrugs and their development has exponentially grown in recent years. In this manuscript, we describe our efforts towards the synthesis of new metallodrugs by reaction of bis(pyridyl)allenes and metal complexes. Two classes of compounds are presented: one in which the allene structure is intact and the metal (Pd(II), Pt(IV) or Au(III)) coordinates to the pyridine-nitrogens; and another, in which one of the pyridines cyclises into a gold-activated allene to form β-N-stabilised gold carbenes. Both classes of compounds are active catalysts in important organic reactions, and are also promising antimicrobial, antifungal and anticancer agents. In this work, we describe the promising anticancer activity, against breast cancer cells, of the gold carbene complexes, and preliminary studies of their interaction with DNA, including non-canonical DNA structures. Our results have revealed an unusual selective stabilisation of hTeloC i-motif by one of the Au(III) carbene complexes, that opens up exciting opportunities for further development of novel DNA-binding metallodrugs

Prediction of DNA i-motifs via machine learning

i-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. The programme iM-Seeker incorporates a Balanced Random Forest classifier trained on genome-wide iMab antibody-based CUT&Tag sequencing data to predict the folding status and an Extreme Gradient Boosting regressor to estimate the folding strength according to both literature biophysical data and our in-house biophysical experiments. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination (R2) of 0.642 on the test set. Model interpretation confirms that the nucleotide composition of the C-rich sequence significantly affects iM stability, with a positive correlation with sequences containing cytosine and thymine and a negative correlation with guanine and adenine

Ligand-induced unfolding mechanism of an RNA G-quadruplex

The cationic porphyrin, TMPyP4, is a well-established DNA G-quadruplex (G4) binding ligand that can stabilize different topologies via multiple binding modes. However, TMPyP4 has completely opposite destabilizing and unwinding effect on RNA G4 structures. The structural mechanisms that mediate RNA G4 unfolding remains unknown. Here, we report on the TMPyP4-induced RNA G4 unfolding mechanism studied by well-tempered metadynamics (WT-MetaD) with supporting biophysical experiments. The simulations predict a two-state mechanism of TMPyP4 interaction via a groove-bound and a top-face bound conformation. The dynamics of TMPyP4 stacking on the top tetrad disrupts Hoogsteen H-bonds between guanine bases resulting in the consecutive TMPyP4 intercalation from top-to-bottom G-tetrads. The results reveal a striking correlation between computational and experimental approaches and validate WT-MetaD simulations as a powerful tool for studying RNA G4-ligand interactions

Replication-induced DNA secondary structures drive fork uncoupling and breakage

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress

Breaking the Access to Education Barrier: Enhancing HPLC Learning with Virtual Reality

This research focuses on an innovative approach to the practical teaching of High Performance Liquid Chromatography (HPLC), specifically exploring the application of Virtual Reality (VR) in undergraduate education. Traditionally, the exposure to HPLC instrumentation for undergraduates has been limited due to a substantial student population and the prohibitively high costs of these systems. To overcome these challenges, we developed our own in-house multi-user VR software, as well as a VR digital twin model of HPLC instruments in our laboratory and placed multiple copies of these in a training environment, aiming to simulate a realistic, interactive, and immersive learning HPLC environment. The investigation of its effectiveness included a group of first year undergraduate students with no previous HPLC experience, aiming to assess the reception of the VR learning environment among a student cohort. The use of the VR software positively influenced student engagement with HPLC training. Survey results indicate that the majority of students greatly enjoyed the VR sessions, with many students reporting a heightened interest in practicals and self-reporting that they learned better than they would have using text or PowerPoints, though formal assessment is needed to quantify its impact on learning outcomes. Notably, students reported a heightened confidence in their operational understanding of the instrument and exhibited a more profound grasp of the underlying theoretical concepts. In light of these findings, we propose that VR learning environments equipped with digital twins of laboratory equipment can greatly enhance practical teaching, particularly in areas constrained by equipment accessibility. This work, therefore, offers compelling insights into the potential of VR learning environments in reshaping HPLC practical teaching in undergraduate education

Redox-dependent control of i-Motif DNA structure using copper cations

Previous computational studies have shown that Cu+ can act as a substitute for H+ to support formation of cytosine (C) dimers with similar conformation to the hemi-protonated base pair found in i-motif DNA. Through a range of biophysical methods, we provide experimental evidence to support the hypothesis that Cu+ can mediate C–C base pairing in i-motif DNA and preserve i-motif structure. These effects can be reversed using a metal chelator, or exposure to ambient oxygen in the air that drives oxidation of Cu+ to Cu2+, a comparatively weak ligand. Herein, we present a dynamic and redox-sensitive system for conformational control of an i-motif forming DNA sequence in response to copper cations

Stability and context of intercalated motifs (i-motifs) for biological applications

DNA is naturally dynamic and can self-assemble into alternative secondary structures including the intercalated motif (i-motif), a four-stranded structure formed in cytosine-rich DNA sequences. Until recently, i-motifs were thought to be unstable in physiological cellular environments. Studies demonstrating their existence in the human genome and role in gene regulation are now shining light on their biological relevance. Herein, we review the effects of epigenetic modifications on i-motif structure and stability, and biological factors that affect i-motif formation within cells. Furthermore, we highlight recent progress in targeting i-motifs with structure-specific ligands for biotechnology and therapeutic purposes

Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Funder: Lister Institute of Preventive Medicine (Lister Institute); doi: http://dx.doi.org/10.13039/501100001255Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress