DNA Hybridization Research Articles

Evaluation of a next generation sequencing assay for Hepatitis B antiviral drug resistance on the oxford nanopore system.

Next-generation sequencing (NGS) for Hepatitis B virus (HBV) antiviral resistance (AVR) testing is a highly sensitive diagnostic method, able to detect low-level mutant subpopulations. Our clinical virology laboratory previously transitioned from DNA hybridization (INNO-LiPA) to NGS, initially with the GS Junior System and subsequently the MiSeq. The Oxford Nanopore Technology (ONT) sequencing system was evaluated for HBV resistance testing, with regards to sequencing accuracy and turn-around time. We performed amplicon sequencing of the HBV polymerase gene from patient plasma and external quality assessment (EQA) samples on the MiSeq Reagent Nano Kit v2 and GridION ONT with R10.4.1 flowcells. Mutational analysis and genotyping were performed by DeepChek®Assay-HBV (version 2.0). A total of 49 patient samples and 15 EQA samples were tested on both the MiSeq and ONT. There was high agreement for both patient and EQA samples between the MiSeq and ONT systems, with regards to total drug resistance mutations detected and total patient sample agreement, 68/70 (97 %) and 47/49 (96 %), respectively. The ONT NGS platform provided accurate HBV AVR results, with improved turn-around times. Sequencing error rates at AVR codons were below 1 %.

P53 mediated regulation of LINE1 retrotransposon derived R-loops.

Long Interspersed Nuclear Element 1 (LINE1/L1) retrotransposons, which comprise 17% of the human genome, typically remain inactive in healthy somatic cells but are reactivated in several cancers. We previously demonstrated that p53 silences L1 transposons in human somatic cells, potentially acting as a tumor-suppressive mechanism. However, the precise molecular mechanisms underlying p53-mediated repression of L1 and its life cycle intermediates remain unclear. In this study, we used DRIP-sequencing experiments to investigate RNA-DNA hybrids, which are key intermediates formed during L1 retrotransposition. Our findings reveal that L1 mRNA-genomic DNA (cis L1 R-loops) and L1 mRNA-complementary DNA (trans L1 R-loops) hybrids, are de-repressed in p53-/- cells. This increase is synergistic with L1 activation by HDAC inhibitors (HDACi). However, treatment with a reverse transcriptase inhibitor reduces this accumulation, indicating that retrotransposition activity plays a significant role in R-loop accumulation. Interestingly, in p53 wild-type cells, hyperactivated L1 transposons are re-silenced upon HDACi withdrawal. L1 resilecing in wt cells coincided with the recruitment of repressive marks, specifically H3K9me3 and H3K27me3, simultaneously preventing the addition of activating marks like H3K4me3, and H3K9ac at the L1 5'UTR. Mechanistically, we demonstrate that p53 cooperates with histone methyltransferases SETDB1 and G9A to deposit H3K9me3 marks at the L1 promoter, thereby silencing transposons. This study is the first to reveal novel roles of p53 in preventing the formation of L1-derived RNA-DNA hybrids (R-loops) and re-silencing of hyperactivated L1 elements by co-operating with histone methyltransferases, underscoring its critical role in maintaining genomic stability.

Fiber ring laser biosensor based on no-core fibers for label-free DNA biomolecule measurements.

An erbium-doped fiber ring laser based on a single-mode fiber-no-core fiber-single-mode fiber (SMF-NCF-SMF) structure was constructed and experimentally demonstrated for label-free DNA hybridization measurement. The SMF-NCF-SMF structure acts as a sensing element and a filter to select the laser wavelength. The proposed fiber ring laser sensor exhibits a high optical signal-to-noise ratio (SNR, >50 dB) and narrow full width at half maximum (FWHM, <0.05 nm). Its refractive index sensitivity is 116.8 nm per RIU in the range of 1.3406-1.3705, and its detection limit is 1.79 × 10-4 RIU. By continuously monitoring the laser wavelength, we successfully achieved label-free measurement of complementary DNA (cDNA) at concentrations as low as 1 μM. Subsequently, the specificity of the sensor was detected by non-complementary DNA (N-cDNA). Experimental results show that the fiber ring laser biosensor has the advantages of simple operation, label-free measurement, and high specificity. Furthermore, it shows a broad application prospect in several fields, especially in key areas such as medical diagnosis and cancer screening.

Reducing Measurement Deviation by Metastable DNA Probes for Aptamer Thermodynamic Characterization.

DNA reaction equilibrium-based calculations have great potential in thermodynamic characterization, but their widespread applications are hindered by significant measurement deviation of equilibrium concentration. Here, we report the advantages of metastable DNA hybridization in reducing quantification deviation of equilibrium concentration and propose a universal and standardized strategy for measuring aptamer binding energy, termed metastable DNA reference calorimetry (MDRC). We built different MDRC-based algorithms tailored to different aptamer binding models, enabling the calculation of thermodynamic parameters for aptamers with one or more binding sites. Our correlative model, considering the cross-effects between different binding sites, showed that for ATP aptamers with two binding sites, binding of the first ATP molecule would decrease its affinity for the second at low temperatures and even completely inhibit this binding at high temperatures. Moreover, the thermodynamic parameters of protein-specific aptamers were calculated to elucidate the universality of the method. The successful analysis of cell-specific aptamers further demonstrated MDRC's applicability in complex biological systems.

FlowBack: A Generalized Flow-Matching Approach for Biomolecular Backmapping.

Coarse-grained models have become ubiquitous in biomolecular modeling tasks aimed at studying slow dynamical processes such as protein folding and DNA hybridization. These models can considerably accelerate sampling but it remains challenging to accurately and efficiently restore all-atom detail to the coarse-grained trajectory, which can be vital for detailed understanding of molecular mechanisms and calculation of observables contingent on all-atom coordinates. In this work, we introduce FlowBack as a deep generative model employing a flow-matching objective to map samples from a coarse-grained prior distribution to an all-atom data distribution. We construct our prior distribution to be agnostic to the coarse-grained map and molecular type. A protein-specific model trained on ∼65k structures from the Protein Data Bank achieves state-of-the-art performance on structural metrics compared to previous generative and rules-based approaches in applications to static PDB structures, all-atom simulations of fast-folding proteins, and coarse-grained trajectories generated by a machine-learned force field. A DNA-protein model trained on ∼1.5k DNA-protein complexes achieves excellent reconstruction and generative capabilities on static DNA-protein complexes from the Protein Data Bank as well as on out-of-distribution coarse-grained dynamical simulations of DNA-protein complexation. FlowBack offers an accurate, efficient, and easy-to-use tool to recover all-atom structures from coarse-grained molecular simulations with higher robustness and fewer steric clashes than previous approaches. We make FlowBack freely available to the community as an open source Python package.

Four novel nontuberculous mycobacteria species: Mycobacterium wendilense sp. nov., Mycobacterium burgundiense sp. nov., Mycobacterium kokjensenii sp. nov. and Mycobacterium holstebronense sp. nov. revived from a historical Danish strain collection.

Four novel nontuberculous mycobacteria were discovered from a historical strain collection at the International Reference Laboratory of Mycobacteriology at Statens Serum Institut in Copenhagen, Denmark. Phylogenetic analysis combining the 16S rrs, internal transcribed spacer and 23S rrl elements, as well as a single-copy core-gene (hsp65, rpoB+C, secA and tuf) analysis of these freeze-dried mycobacteria, clinically isolated from gastric lavage samples between 1948 and 1955, showed to be associated with type strains grouping within the Terra and Fortuitum-Vaccae clade. Phenotypic characteristics, biochemical properties and fatty acid and mycolic acid profiles supported the classification as novel strains. A genomic comparison to the closest related type strain was done by calculating average nucleotide identity and in silico DNA:DNA hybridization values, which showed 87.9% and 33.0% for Mu0050, 85.2% and 27.4% for Mu0053, 85.3% and 27.6% for Mu0083 and 93.3% and 50.1% for Mu0102, respectively. The names proposed for the new species are Mycobacterium wendilense sp. nov. (Mu0050T=ITM 501390T=CCUG 77525T), Mycobacterium burgundiense sp. nov. (Mu0053T=ITM 501391T=CCUG 77526T), Mycobacterium kokjensenii sp. nov. (Mu0083T=ITM 501392T=CCUG 77527T) and Mycobacterium holstebronense sp. nov. (Mu0102T=ITM 501393T=CCUG 77528T).

DNA nanotechnology-based strategies for minimising hybridisation-dependent off-target effects in oligonucleotide therapies.

Targeted therapy has emerged as a transformative breakthrough in modern medicine. Oligonucleotide drugs, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have made significant advancements in targeted therapy. Other oligonucleotide-based therapeutics like clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are also leading a revolution in targeted gene therapy. However, hybridisation-dependent off-target effects, arising from imperfect base pairing, remain a significant and growing concern for the clinical translation of oligonucleotide-based therapeutics. These mismatches in base pairing can lead to unintended steric blocking or cleavage events in non-pathological genes, affecting the efficacy and safety of the oligonucleotide drugs. In this review, we examine recent developments in oligonucleotide-based targeted therapeutics, explore the factors influencing sequence-dependent targeting specificity, and discuss the current approaches employed to reduce the off-target side effects. The existing strategies, such as chemical modifications and oligonucleotide length optimisation, often require a trade-off between specificity and binding affinity. To further address the challenge of hybridisation-dependent off-target effects, we discuss DNA nanotechnology-based strategies that leverage the collaborative effects of nucleic acid assembly in the design of oligonucleotide-based therapies. In DNA nanotechnology, collaborative effects refer to the cooperative interactions between individual strands or nanostructures, where multiple bindings result in more stable and specific hybridisation behaviour. By requiring multiple complementary interactions to occur simultaneously, the likelihood of unintended partially complementary binding events in nucleic acid hybridisation should be reduced. And thus, with the aid of collaborative effects, DNA nanotechnology has great promise in achieving both high binding affinity and high specificity to minimise the hybridisation-dependent off-target effects of oligonucleotide-based therapeutics.

Towards melanoma in situ vaccination with multiple ultra-narrow X-ray beams.

Despite the recent progress, current treatment modalities are not able to eradicate cancer. We show that Microbeam Radiotherapy (MRT), an innovative type of Spatially Fractionated Radiotherapy, can control murine melanoma by activating the host's own immune system. The beneficial effects are very pronounced in comparison to uniform radiotherapy traditionally employed in the clinic. Our results show that MRT increased antigen presentation, activating Cytotoxic T Lymphocytes (CTLs) which are essential to MRT's treatment efficacy in melanoma. Depletion of CTLs abrogated treatment response. Multiplex nucleic acid hybridization technology revealed key features of lymphocyte populations such as proliferation, differentiation, and ligand-receptor interactions. In addition, CTLs were shown to be essential for locoregional metastatic control and systemic abscopal effects confirmed by activation of antigen presenting cells and CTL trafficking in the tumour-draining lymph nodes. MRT also showed a synergistic effect with immunotherapy. Overall, MRT induces a robust antitumour immune response, acting like an in situ vaccination, which could be exploited to treat a variety of treatment-resistant malignancies.

Enzyme fragment complementation driven by nucleic acid hybridization sans self-labeling protein.

A modified enzyme fragment complementation assay has been designed and validated as a turn-on biosensor for nucleic acid detection in dilute aqueous solution. The assay is target sequence-agonistic and uses fragments of NanoBiT, the split luciferase reporter enzyme, that are esterified enzymatically at their C-termini to steramers, sterol-linked oligonucleotides. The Drosophila hedgehog autoprocessing domain, DHhC, serves as the self-cleaving enzyme for the NanoBiT-steramer bioconjugations. Unlike current approaches, the final bioconjugate generated by DHhC and used for nucleic acid detection is free of self-labeling passenger protein. In the presence of single stranded (ss) DNA or RNA template with adjacent segments complementary to the Nano-BiT steramer oligonucleotides, the two NanoBiT fragments associate productively, reconstituting NanoBiT's luciferase activity. In samples containing ssDNA or RNA template at low nM concentrations, NanoBiT luminescence exceeded background signal by 30- to 60-fold. The steramer probe sequences used to prepare these sensors are unconstrained in length and composition. In the absence of sequence constraints of the probe element and without the added bulk of a self-labeling protein, these NanoBiT-steramer bioconjugates open new applications in the programmable detection of small fragments of coding and noncoding DNA and RNA.

Light harvesting FIT DNA hybridization probes for brightness-enhanced RNA detection.

Fluorogenic hybridization probes are essential tools in modern molecular biology techniques. They allow detection of specific nucleic acid molecules without the need to separate target-bound from unbound probes. To enable detection of targets at low concentration, fluorogenic probes should have high brightness. Here, we report the development of RNA hybridization probes (RNA FIT probes) that use smart quenching and a light harvesting principle to enhance the brightness of fluorescence signaling. The signaling mechanism is based on FRET between brightly emitting donor dyes and a fluorescent base surrogate, such as quinoline blue (QB) or thiazole orange (TO). In the single-stranded state, QB/TO nucleotides fluoresce weakly and quench the fluorescence of the donor dyes. Upon target recognition, QB/TO stack with adjacent base pairs, resulting in enhanced fluorescence quantum yields. The donor dyes are blue-shifted by only 5-20 nm relative to the QB/TO nucleotides, allowing simultaneous excitation of both dye groups with efficient energy transfer. The combined photon absorption results in exceptionally bright FIT probes. This feature facilitated the detection of RNA target in undiluted cell lysates. The present study examines the utilization of probes to detect mRNA targets in live T cells using flow cytometry.

Characterization of boron doped diamond electrodes with engineered sp2 carbon content and their application to structure-dependent DNA hybridization

Characterization of boron doped diamond electrodes with engineered sp2 carbon content and their application to structure-dependent DNA hybridization

DNA-amphiphilic nanostructures: synthesis, characterization and applications.

DNA's extraordinary potential reaches far beyond its role as a carrier of genetic information. It serves as a remarkably adaptable structural foundation for constructing intricate nanostructures with a diverse range of functionalities. This inherent programmability sets DNA apart from other biomolecules like peptides, proteins, and small molecules. By covalently attaching DNA to synthetic hydrophobic moieties, researchers create DNA amphiphiles capable of interacting with artificial lipid bilayers and cell membranes. These hybrid structures have rapidly gained prominence due to their promising potential in the medical field. This review provides a comprehensive overview of the latest advancements in the synthesis of DNA amphiphiles and their assembly into well-defined nanostructures. It explores the diverse applications of these nanostructures across various medical domains, including targeted drug delivery, innovative immunotherapies, and gene-silencing techniques. Moreover, the review delves into the current challenges and prospects of this rapidly evolving field, highlighting the potential of DNA hybrid materials to revolutionize medical treatments and diagnostics. By addressing the limitations and exploring new avenues of research, scientists aim to unlock the full potential of DNA nanotechnology for the benefit of human health.

DNA Dendron Tagging as a Universal Way to Deliver Proteins to Cells.

The use of proteins as intracellular probes and therapeutic tools is often limited by poor intracellular delivery. One approach to enabling intracellular protein delivery is to transform proteins into spherical nucleic acid (proSNA) nanoconstructs, with surfaces chemically modified with a dense shell of radially oriented DNA that can engage with cell-surface receptors that facilitate endocytosis. However, proteins often have a limited number of available reactive surface residues for DNA conjugation such that the extent of DNA loading and cellular uptake is restricted. Indeed, DNA surface density and sequence have been correlated with scavenger-receptor engagement, the first step of cellular internalization. Here, we report how branched DNA dendrons with dibenzocyclooctyne groups and proteins genetically engineered to include the noncanonical amino acid azido-phenylalanine for click chemistry can be used to synthesize hybrid DNA dendron-protein architectures that exhibit outstanding cellular internalization properties, without the need for extensive surface modification. In a head-to-head comparison, protein-DNA dendron structures (where DNA is concentrated in a local area) are taken up by cells more rapidly and to a greater extent than proSNAs (where the DNA is evenly distributed). Also, protein-G-rich dendron structures show enhanced uptake compared to protein-T-rich dendron structures, highlighting the importance of oligonucleotide sequence on nanoconjugate uptake. Finally, a generalizable method for chemically tagging proteins with dendrons that does not require mutagenesis is described. When a range of proteins, spanning 42 to 464 kDa, were modified through surface lysines with this method, a significant increase in their cellular uptake (up to 17-fold) compared to proteins that are not coupled to a DNA dendron was observed.

Advancing Genetic Disorder Detection: Integrating Nanomaterial-Enhanced Biosensors with Genetic Markers

Genetic disorders pose significant challenges in health care because of the more nuanced and costly methods that prolong patient treatment and recovery times. In addition, to ensure a more accurate diagnosis, the process for the detection of this disease can be quite complex and requires excessive testing and counseling, such as hereditary testing. Given these challenges, a new option has arisen in recent decades: nanomaterial-enhanced biosensors. These diagnostic methods are cutting edge and stand out from traditional methods in many areas, some of which include sensitivity, selectivity, and stability. Among other nanomaterials, gold nanoparticles (AUNPs), carbon nanotubes (CNTs), graphene (GR), and quantum dots (QDs) stand out in terms of their performance metrics and are the most promising for the field. Thus, they are examined in depth in this paper. AUNPs enable visible color changes for genetic disorder detection through DNA hybridization, CNTs have high sensitivity and conductivity for detecting genetic mutations, GR provides exceptional electrical conductivity and biocompatibility for various biosensing utilizations, and QDs allow for precise and accurate diagnostics owing to their light-emitting properties. Nanomaterial biosensor technology has greatly improved in the field of genetics, including innovations in wireless technology and advancements in faster and more specific disease detection. These findings have helped significantly improve the diagnosis of health conditions. Even with all of these benefits, there are still issues with sensitivity, stability, biocompatibility, nonspecific binding, a low dynamic range, and high costs. Other major obstacles are ethical concerns, such as data privacy. Nanomaterial-based biosensors undoubtedly can change the game in regard to diagnosing and treating genetic conditions, but additional research must be done to improve the cost, applicability, and reliability.

Tuning the Duplex Stability of DNA Oligonucleotides Containing Metal-Mediated Base Pairs of Imidazole-Derived Nucleobases.

Two artificial imidazole-derived nucleobases, HQIm (3H-imidazo[4,5-f]quinolin-5-ol) and CaIm (imidazole-4-carboxylate), were introduced into short DNA duplexes to systematically investigate their thermal stability upon metal ion coordination. Metal-mediated base pairs are formed with the 3d metal ions CoII, NiII and ZnII, as well as with the lanthanoid ions EuIII and SmIII, which induce a thermal stabilization of up to 8 °C upon binding. The latter are the first lanthanoid-mediated base pairs involving only four donor atoms that result in a significant duplex stabilization. For the 3d metal ions, the increase in DNA melting temperature is in agreement with the trend in complex stability according to the Irving-Williams series. The duplex stabilization upon metal-mediated base pair formation depends on the identity of the artificial base pair, enabling discrimination between metal ions by systematic design of the duplex. Further, the influence of pH on the stability of selected duplexes was studied, revealing a linear relation between pH and the formation of HQIm-NiII-HQIm base pairs and allowing to induce a distinct stabilization between 1 °C and 28 °C. Regarding EuIII-mediated base pairs, an acidic pH can be utilized to disturb their formation, presumably by promoting the coordination of EuIII to the DNA backbone.

Transcription as a double-edged sword in genome maintenance.

Genome maintenance is essential for the integrity of the genetic blueprint, of which only a small fraction is transcribed in higher eukaryotes. DNA lesions occurring in the transcribed genome trigger transcription pausing and transcription-coupled DNA repair. There are two major transcription-coupled DNA repair pathways. The transcription-coupled nucleotide excision repair (TC-NER) pathway has been well studied for decades, while the transcription-coupled homologous recombination repair (TC-HR) pathway has recently gained attention. Importantly, recent studies have uncovered crucial roles of RNA transcripts in TC-HR, opening exciting directions for future research. Transcription also plays pivotal roles in regulating the stability of highly specialized genomic structures such as telomeres, centromeres, and fragile sites. Despite their positive function in genome maintenance, transcription and RNA transcripts can also be the sources of genomic instability, especially when colliding with DNA replication and forming unscheduled pathological RNA:DNA hybrids (R-loops), respectively. Pathological R-loops can result from transcriptional stress, which may be induced by transcription dysregulation. Future investigation into the interplay between transcription and DNA repair will reveal novel molecular bases for genome maintenance and transcriptional stress-associated genomic instability, providing therapeutic targets for human disease intervention.

Visualized Nucleic Acid Hybridization Lateral Flow Strip Integrating with Microneedle for the Point-of-Care Authentication of Ophiocordyceps sinensis.

Due to the price and demand of Ophiocordyceps sinensis having increased dramatically, adulteration with other fungi is a common problem. Thus, a reliable method of authentic O. sinensis identification is essential. In the present work, a rapid DNA extraction and double-tailed recombinase polymerase amplification (RPA) coupled with nucleic acid hybridization lateral flow strip (NAH-LFS) was developed to distinguish authentic O. sinensis ingredients from other fungi substitutes. In the presence of O. sinensis, the RPA amplicons with two ssDNA tails in the opposite ends, which could simultaneously bind with the SH-probes on gold nanoparticles (AuNPs) and capture the probe on the test line, formed visible red bands. RPA combined with NAH-LFS can efficiently detect O. sinensis DNA down to 1.4 ng/μL; meanwhile, the specificity test validated no cross reaction with common adulterants, including Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, yungui Cordyceps, and Ophiocordyceps nutans. The whole RPA-NAH-LFS could be completed within 16 min. The RPA-NAH-LFS results in detecting 20 commercial O. sinensis samples are consistent with PCR-AGE and RT-PCR, confirming the feasibility of the RPA-NAH-LFS method. In conclusion, these results are expected to facilitate the application of RPA-NAH-LFS in the authentication detection of O. sinensis materials, providing a convenient and efficient method for O. sinensis quality control.

Impact of Surface Adsorption on DNA Structure and Stability: Implications for Environmental DNA Interactions with Iron Oxide Surfaces.

Environmental DNA (eDNA), i.e., DNA found in the environment, can interact with various geochemical surfaces, yet little is known about these interactions. Mineral surfaces may alter the structure, stability, and reactivity of eDNA, impacting the cycling of genetic information and the reliability of eDNA-based detection tools. Understanding how eDNA interacts with surfaces is crucial for predicting its fate in the environment. In this study, we examined the surface interaction and stability of herring testes DNA, a model system for eDNA, on two common iron oxide phases present in the environment: α-FeOOH (goethite) and α-Fe2O3 (hematite). Utilizing spectroscopic probes, including attenuated total reflection Fourier-transform infrared (ATR-FTIR) and UV-vis spectroscopy, we quantified the DNA adsorption capacity at pH 5 and determined its secondary structure. DNA adsorbed irreversibly at pH 5 and 25 °C, primarily through its phosphate groups, and retained the solution-phase B-form structure. However, the infrared data also indicated some distortion of the B-form likely due to additional interactions between nitrogenous bases when adsorbed on the α-Fe2O3 particle surfaces. The distortion in the double helical structure of adsorbed DNA on α-Fe2O3 led to a lower melting temperature (Tm) of 60 °C compared to 70 °C for DNA in solution. In contrast, DNA adsorbed on α-FeOOH melted at higher temperatures relative to solution-phase DNA and in two distinct phases. Upon testing adsorbed DNA stability at higher pH values, there were distinct differences between the two iron oxide phases. For α-FeOOH, nearly 50% of the DNA desorbed from the surface when the solution pH changed from 5 to 8, while less than 5% desorbed from α-Fe2O3 under the same conditions. Overall, these findings underscore the importance of mineral-specific eDNA-surface interactions and their role in adsorbed eDNA stability, in terms of DNA melting and the impact of solution-phase pH changes.

Probe-Type High-Sensitivity DNA Hybridization Detection Based on Optical Fiber SPR

Probe-Type High-Sensitivity DNA Hybridization Detection Based on Optical Fiber SPR

Unraveling the Molecular Basis for G-Quadruplex-Binders to ALS/FTD-Associated G4C2 Repeats of the C9orf72 Gene.

The most recurrent familial cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the presence of an abnormal number of intronic GGGGCC (G4C2) repetitions in the C9orf72 gene, which has been proposed to drive ALS/FTD pathogenesis. Recently, it has been shown that such G4C2 repetitions can fold into G-quadruplex (G4) secondary structures. These G4s have been selectively stabilized by small-molecule binders, furnishing proof-of-principle that targeting these non-canonical nucleic acid sequences represents a novel and effective therapeutic strategy to tackle neurodegenerative disorders. However, precise information on the mechanism of action of these compounds is still lacking. Here, by performing in silico investigations, we unraveled the molecular basis for the selectivity of a series of known structurally related C9orf72 G4-binders. Moreover, we investigated the binding properties of a strong and selective metal-based G4 stabilizer, the AuI bis-N-heterocyclic carbene (NHC) complex - Au(TMX)2 - showing that it moderately stabilizes G4C2 G4 RNA by Förster resonance energy transfer (FRET) DNA melting assays. Using metadynamics (metaD) simulations, the Au(TMX)2 binding mode and the associated free-energy landscape were also evaluated. This information paves the way for developing improved compounds to tackle ALS/FTD neurodegenerative disorders.