Invited Speakers

The development of selective ligands to target DNA G-quadruplexes (G4s) has been pivotal in supporting their role in transcriptional regulation.¹ However, most of the ligands described to date lack intra-G4 selectivity, severely limiting their potential for uncovering the biological function of individual G4s across the genome. In this talk, I will present ATENA (Approach to Target Exact Nucleic Acid alternative structures) a new method we developed to provide G4-ligand with intra-G4 selectivity. ATENA relies on the chemical modification of established G4-ligands that can be conjugated to a catalytically inactive Cas9 protein (dCas9) using Halo-Tag, enabling the targeting of individual G4s in living cells. We have systematically screened the length of the PEG-linkers connecting the G4-ligands to the Halo-Tag and sgRNA sequences to attain optimal G4-engagement both in vitro and in cells. Using optimised conditions, we leveraged ATENA to demonstrate how selective targeting of the established G4 in the proto-oncogene c-MYC suppresses its transcription exclusively from the P1 promoter. We also show that positioning ligands in the proximity of TATA-boxes suppresses c-MYC transcription in a G4-independent manner, highlighting the importance of appropriate design to measure real G4-mediated transcriptional changes. We also demonstrate that selective targeting of the PVT-1 G4 by conjugation of dCas9 to different G4-ligands, PDS or PhenDC3, leads to either stimulation or repression of PVT-1 transcription, indicating that the biological responses elicited by G4-stabilization can be highly dependent on the type of ligand used. We further leveraged our platform to study the biological responses caused by targeting de novo G4s. Our study provides key insights into G4-based therapeutic design, offering an innovative platform to investigate G4 biology from a fresh perspective.

G-quadruplex (G4) DNA structures are dynamic epigenetic phenomena that form in nucleosome-depleted regions (NDRs) (e.g., promoters, enhancers).Active enhancers are marked by H3K27ac, crucial for gene regulation. Innate immune memory (trained immunity) is another epigenetic event in which immune cells (e.g., macrophages) recognize and “memorize” exogenous signals in their chromatin, leading to enhanced immune responsiveness in the future. We hypothesize that G4s, which predominate in NDRs and exclude nucleosomes, support long-lasting transcription of immune genes critical for trained immunity. Indeed, we observe persistent H3K27ac near G4-forming sites in monocyte-derived macrophages from human individuals who received ≥2 SARS-CoV-2 mRNA vaccinations, suggesting that G4 structures enhance immune memory.G4s can promote DNA double-strand breaks (DSBs) when exogenously stabilized by small molecules targeting G4s and are thought to cause mutations when not properly maintained by helicases. Previously, we used G4-mapping to reveal their increased formation in various cancer models, suggesting that their presence in regulatory and unstable regions may contribute to cancer development. I will present our efforts to quantify G4 levels in human and murine aging models and how the loss of sirtuin function may explain increased G4 levels, persistent DSBs, and senescence in aged human and murine cells as well as in aged tissues.

The stability and normal expression of mtDNA are essential for cellular respiration and tissue viability. Secondary structures are present in mtDNA, but we have an incomplete understanding of how they impact mitochondrial replication, gene expression, and genome integrity. We and others have associated G-quadruplex structures (G4s), which form among guanine-rich sequences, with mtDNA deletion breakpoints. Our exciting preliminary data shows that mitochondrial-localized G4 binding agents (mtG4BAs) negatively influence mitochondrial replication, transcription, and respiration through G4 stabilization and have a biased interaction with mtDNA sequence variants that affect G4 stability. Despite this, mitochondrial G4 are evolutionarily conserved, suggesting that G4s have a beneficial role in mitochondrial function at physiological levels. These data lead us to the overarching hypothesis that physiological G4 formation within mtDNA regulates mitochondrial transcription and replication. The mtDNA is rich in predicted G4-forming sequences, but it is unclear which mitochondrial sequences usually form G4 in cells, how these structures affect mitochondrial biology, and which specific enzymes regulate G4 formation and removal. We will describe what we currently know about mtG4 formation, biological impact, and tools being generated for their study. The hope is that mtG4 can be leveraged with targeting molecules to treat mitochondrial defects in genetic and idiopathic diseases.

DNA secondary structure and topology are in dynamic flux. Important question is what is the functional significance of non-canonical DNA structures. Sequences containing runs of guanines and capable of forming stable G4 DNA are highly enriched at gene promoters suggesting their potential role in regulating transcription. Recently, human Zn-finger transcription factors such as Sp1 and YY1 were shown to be G4 DNA-binding proteins. Our preliminary experiments led to the novel discovery that yeast transcription factor Msn2 binds to G4 DNA in vivo and in vitro. And G4 DNA forming sequences (PQS) are present at many of Msn2-target gene promoters. Transcription at these genes are up-regulated by G4 ligands. Here, we describe the kinetics and specificities of Msn2 interaction with G4 DNA and the effect of Msn2-G4 interaction in the regulation of transcription. We identified additional Zn-Finger transcription factors in both yeast and human that bind to G4 DNA with high affinity in support of a novel mode of transactivation in which G4 DNA becomes a docking site for multiple TFs including Msn2 to establish a hub of transactivation.

A common feature of most viruses is their dependence on regulatory RNA elements. Recent research suggested that non-canonical structures called G-quadruplexes (G4s) are overrepresented in viral genomes and have emerged as promising anti-viral targets. Using yellow fever virus (YFV) as a model system, we characterized the formation and the biological consequences of a conserved G4 within the genomes of the Flaviviridae family. We determined that this G4 is essential to promote viral replication and suppress the host cell stress response pathway. In subsequent mechanistic analyses, we pinpoint that this unique G4 function is associated with the nuclear host protein hnRNPH1. Specifically, G4 interaction leads to the retention of hnRNPH1 in the cytoplasm, causing an impaired stress response and alleviation of the anti-viral effects of stress-induced G4-forming tRNA fragments (tiRNAs). In conclusion, our data reveal a unique interplay of hnRNPH1 with host and viral G4 targets, controlling the integrated stress response and viral infection.

Guanine (G)-rich sequences in nucleic acids can form noncanonical secondary structures such as R-loop and G-quadruplex (G4) during transcription. The R-loop formed on the template strand promotes and stabilizes G4 in the non-template strand. However, the precise role of G4/R-loop-forming sequences on transcription remains poorly understood. In this study, we investigated the effect of different potential G4-forming sequences (PQS) on G4/R-loop formation and transcription dynamics. We employed gel-based assays and single-molecule fluorescence resonance energy transfer (FRET) to measure RNA synthesis and concomitant formation of G4 and R-loop during transcription by T7 RNA polymerase. We reveal two types of R-loop that form successively; an R-loop with an intramolecular DNA G4 (IG4) initially forms during transcription, followed by an R-loop with an intermolecular DNA: RNA hybrid G4 (HG4). We found that IG4 R-loops inhibit whereas HG4 R-loops enhance transcription. We identified that an HG4/IG4 ratio highly correlates with transcriptional activity. PQS with short linkers favors IG4, reducing transcription, while PQS with long linkers that induce loosely folding PQS favor HG4, increasing transcription. Since IG4 formation precedes HG4, tightly folded PQS forms IG4 quickly and stably, slowing its conversion to HG4 and reducing transcriptional enhancement.

G-quadruplexes (G4s) can recruit transcription factors to activate gene expression, but detailed mechanisms are not well characterized. We demonstrate that G4s in the CCND1 promoter promote the molecular motility in MAZ phase-separated condensates and subsequently activate CCND1 transcription. Zinc finger (ZF) 2 of MAZ is responsible for G4 binding, while ZF3-5, but not a highly disordered region, is critical for MAZ condensation. MAZ nuclear puncta overlaps with signals of G4s and various coactivators including BRD4, MED1, CDK9 and active RNA polymerase II, as well as gene activation histone markers. MAZ mutants lacking either G4 binding or phase separation ability did not form nuclear puncta, and showed deficiencies in promoting hepatocellular carcinoma cell proliferation and xenograft tumor formation. Overall, we unveiled a novel mechanism that G4s recruit MAZ to the CCND1 promoter and facilitate the motility in MAZ condensates that compartmentalize coactivators to activate CCND1 expression and subsequently exacerbate hepatocarcinogenesis.

Recent studies documented the widespread presence of guanine quadruplex (G4) in the human genome and transcriptome. Identifying cellular proteins involved in the recognition of G4 structures is important for understanding the biological functions of G4.  In this presentation, I will discuss our recent experimental and bioinformatic approaches toward identifying novel DNA and RNA G4-binding proteins in vitro and in live cells. Affinity pull-down, photo-crosslinking and proximity labeling, followed by quantitative proteomic analysis, allowed us to identify a large number of candidate DNA and RNA G4-binding proteins. Bioinformatic analysis of publicly available ChIP-seq and CLIP-seq data also resulted in the identification of candidate G4-binding proteins. We also validated the abilities of some of these proteins in binding directly and selectively with G4 DNA and RNA. Moreover, we illustrated the functions of these newly identified G4-binding proteins in modulating various important biological processes, including long-range DNA looping, mRNA splicing, alternative polyadenylation, and telomere maintenance.  Together, our integrative chemical biology approach provided a better understanding about the biological functions of DNA and RNA G4.

Liquid-liquid phase separation (LLPS) occurred inside cells has attributed to modulating a multitude of cellular functions. These LLPS condensates are often formed between positively charged peptides and negatively charged nucleic acids. In our research, we have investigated the LLPS condensation between peptides and single-molecule nucleic acid templates anchored between two optically trapped beads in a laser tweezers instrument. By monitoring the mechanical tension of the nucleic acid templates upon addition of positively charged poly-l-lysine (PLL), we revealed a multistage LLPS process mediated by the long-range interactions between nucleic acids and polyelectrolytes. We found that compared to random nucleic acid sequences, single-stranded DNA templates containing tandem G-quadruplex units are most difficult to form LLPS condensates. This is likely because poly-G-quadruplex has the propensity to form rigid nucleic acid-PLL complexes, reducing the condensate formation during the LLPS process. In addition, we also discovered that LLPS processes follow matched chirality between poly-lysine and G-quadruplex. Poly-l-lysine and poly-d-lysine are easier to form condensates with left- and right-handed G-quadruplexes, respectively. We anticipate that these results can provide new strategies to interfere with biological functions of LLPS condensates involving nucleic acids with secondary structures, such as tandem G-quadruplex, which occurs frequently inside cells.

The pUG fold is an unusual left-handed (Z-form) quadruplex that forms in poly(UG)-rich RNA sequences.  We determined the high-resolution structure of the pUG fold and showed it is responsible for directing the amplification of RNA interference. We discovered that pUG folds mark mRNAs as vectors for the transgenerational epigenetic inheritance of gene silencing in vivo. We used several orthogonal methods, including atomic-level mutagenesis of reporter RNAs containing 7-deazaguanosine, to demonstrate this. The injection of atomically modified RNAs into live animals, accompanied by measurement of the silencing efficiency of GFP reporters, allowed us to provide direct evidence for functional importance of the pUG fold. I will describe the unusual biophysical properties of pUG RNAs, including their structure, dynamics and ability to form high-order multimeric interactions that enable efficient formation of condensates. I will also describe the “rules” that govern pUG folding and will show many sequences that are not poly(UG) can still adopt this unusual fold. These data lead to the identification of many thousands of potential pUG folds in regulatory regions of human RNAs, and some of these are associated with disease.  The rules for understanding pUG folding broaden our understanding of nucleic acid quadruplexes and will be important in the future for understanding when and where pUG folds form within transcriptomes.

Prion protein (PrP) causes prion diseases. PrP also functions as a receptor of Aβ protein to transmit the pathological signal of Aβ into cells, resulting in the repression of long-term potentiation (LTP) (Lauren et al., Nature, 2009). We isolated an RNA aptamer, that forms quadruplex, against the PrP, and determined the structure in complex with PrP. This clarified a mechanism how the aptamer exerts high affinity (Mashima et al., NAR, 2013). We also demonstrated spectroscopically that this aptamer inhibits the interaction between Aβ and its receptor, PrP, through tight binding to PrP (Iida et al., FEBS J., 2019). Recently, we have revealed electrophysiologically that the aptamer rescues the LTP repressed by Aβ, through the inhibition of the Aβ-receptor (PrP) interaction (Nakao, et al., in preparation).   We succeeded in observing the in-cell NMR signals of nucleic acids in living human cells for the first time. Then, we found that a G:G base pair of the quadruplex DNA opens more frequently in living cells than in vitro (Yamaoki et al., Nature Commun., 2022; Eladl et al., Chem. Commun., 2022; Eladl et al., Int. J. Mol. Sci., 2023; Eladl et al., submitted).

Investigations into i-motifs in telomeric regions have shown their ability fold into tetrahelical i-motif structures in vitro however little has been revealed structurally to understand their drugability, possibility due to the limited understanding in their role in human telomere biology. Recent data has shown that telomeric i-motifs can inhibit the activity and processivity of telomerase extension, highlighting a possible therapeutic role. Here we report on the use of single based FRET studies on telomeric i-motifs within ssDNA and dsDNA constructs. Sequences were labelled allowing us to follow pH dependent folding/unfolding pathways, both temporal and spatially. The constructs were also designed to show the contribution of the presents of dsDNA, both at the 3’ end and internally. Our results show impact on i-motif stability when in the presence of dsDNA. Crystallisations were also undertaken on these model constructs leading to the successful crystallisations of two of these constructs. Using SAD methods, we have now determined the crystallographic structure of the intramolecular i-motif from the human telomeric sequence. Our overall vision is to reveal the detail in iM structures and their ligand binding sites, enabling the potential of iM structures as therapeutic molecular targets for rational drug design.

Pif1 is a superfamily 1B helicase implicated in unfolding of G-quadruplex DNA (G4s).  G-quadruplex DNA can be formed when guanine-rich nucleic acids induce four-stranded tetrads that are held together by Hoogsteen base pairing and stabilized through stacking interactions. G4s are sufficiently stable to cause genome instability through impeding DNA replication fork progression. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the structural mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5’ single-stranded DNA (ssDNA) segment.  ScPif1 recognizes G4 mainly through a region referred as the “wedge” in the 1A domain that contacts the 5’ most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases.

1. Time-resolved NMR to study the effect of G4 binding ligands on G4 folding. This work has been done by I. Burkhart together with J. Plavez.
2. Time-resolved NMR using photoreversible ligands to study G4 folding. This work has been done by J. Martins.
3. The effect of Zuo-1 protein on a metastable G4 forming sequence from yeast.

This work has been done by I. Burkhart and M.  Limmer together with K. Paeschkie and J. C. Penedo.

Tetraplexes including G-quadruplex and i-motif play a crucial role in gene expression and diseases. As these tetraplexes are highly responsive to the environment changes in solution, molecular environment should have a main factor to control tetraplex formation and functions in cells. Here, we systematically investigated the stability and functions of i-motif DNAs by using various polyethylene glycols that mimicked diverse cellular crowding environments. The thermodynamic and molecular dynamics simulation revealed that the helicity of the i-motif dynamically changed depending on various physicochemical factors of the solution environment. Furthermore, cosolute-induced twisting dynamics controlled by different cosolutes changed the activation energy barrier of replication along the i-motif-forming DNAs. Our findings implied that the regulatory mechanism of the biological roles of i-motifs in different cellular phases does not rely on binding proteins. We will also discuss the function of G-quadruplex under various conditions in cells.

G-quadruplexes (GQs) are noncanonical DNA secondary structures composed of stacks of G-tetrads. They play important biological roles and have therapeutic potential as drug targets. Although many 2-4 G-tetrad GQs have been well characterized, monomolecular sequences with the potential to form 5 or more G-tetrads have not. Here, we investigate sequences with four stretches of 5Gs connected by loops of differing length and capped by 0-4T (labeled LM). Biophysical characterization of 34 LM variants indicates that they fold into predominantly antiparallel GQs—only five variants display significant hybrid/parallel character. PAGE revealed that LM variants are monomolecular and relatively homogeneous. All LM variants display high stability. Increase in the number of 5’-T or loop length diminishes the antiparallel fold and decreases stability of the GQs, with loop length having stronger effect. 3’-T have less effect on the fold and stability. We designed mutants where each 5G stretch was trimmed to 4G – these mutants displayed significantly lower thermal stability (by > 6 °C) suggesting that LM variants indeed contain 5-tetrads. We solved a crystal structure of one of the variants which displayed an interlaced dimer of 5-tetrad hybrid GQs. One G from each monomer participates in the formation of the first G-tetrad in the symmetry generated partner. The 5-tetrad GQs can be great therapeutic targets, as their rarity would lead to a greater selectivity of drugs that target them.

Selective stabilization of individual promoter G-quadruplexes (G4s) offers therapeutic potential for cancer, neurological disorders, and Fragile X syndrome. While achieving high selectivity with small molecules remains challenging, oligonucleotide (ODN) therapeutics can overcome this selectivity hurdle, though they face difficulties with cellular delivery and uptake. Our group pioneered a clamp-based oligonucleotide approach (termed DNAi) to stabilize the MYC promoter G4 by binding flanking DNA with a limited linker. We optimized this DNAi for cost-effectiveness and biological activity in lymphoma. To address limitations in cellular delivery and uptake, this presentation will discuss modifications to the DNAi backbone, coupling to cell-penetrating peptides, and targeted delivery with antibodies. These advances were undertaken in order to develop the ODN therapeutically for mantle cell lymphomas and breast cancer, where we will discuss cellular stability, localization, and biological effects. The DNAi technology has potential applications for a range of therapeutically tractable G4s in oncological, inflammatory, and other critical disease states.

Protein homeostasis governs many diseases, including most neurodegenerative diseases. RNA has long been suspected to directly impact these processes, and it is imperative to determine which RNAs are particularly important in protein folding and aggregation, and their specific effects in biology and pathology. Through a combination of in vitro, cellular, and C. elegans approaches, we determined that G-quadruplexes are powerful modulators of protein folding and aggregation. For example, we found that G-quadruplexes can catalyze protein folding, but under chronic stress, G-quadruplexes can seed protein oligomerization and are a common mechanistic link between disparate neurodegenerative diseases, and that modulating G-quadruplexes is a promising general therapeutic approach for neurodegeneration.

Cancer stands as a pervasive global health challenge and chemotherapy stands as the prevailing approach to treat it. While traditional chemotherapeutic agents have proven effective in cancer treatment, the consequential impact on the physical and psychological well-being of patients remains notably severe. Furthermore, the adaptability of tumor cells to develop resistance against a wide array of chemotherapeutic drugs poses a significant obstacle, ultimately resulting in treatment failures.In this context, chemo-sensitization has gained attention as a strategy using small molecules to enhance cancer cells’ sensitivity to conventional drugs. This approach aims to tackle chemoresistance mechanisms, reduce chemotherapy-induced side effects, and improve clinical outcomes.This presentation explores the growing relevance of G-quadruplex (G4) structures as promising anti-cancer targets. The goal is to boost the antitumor efficacy of standard chemotherapeutics by leveraging G4-interacting molecules, enabling synergistic effects with conventional drugs while minimizing toxicity in healthy cells and overcome drug resistance.