G-rich Regions Research Articles

Primary screening of chemically modified immunosuppressive oligonucleotides using in vitro model with spleen lymphocytes

Control of immune response following transplantation of cells, tissues, or organs includes reduction negative effects caused by acute graft-versus-host disease (GVHD) developing during bone marrow transplantation, thus being an urgent task of modern clinical practice. In this view, the management of immunological tolerance is a promising approach, in particular, the ability of immune cells (especially, dendritic cells) to induce this response using experimental models of allogeneic transplant rejection, GVHD and autoimmune disorders. Therefore, the search for compounds that can effectively activate or suppress immune cells and regulate immunological tolerance is of importance. The purpose of this work was to study the effects of synthetic immunosuppressive oligodeoxynucleotides (INH-ODN) on in vitro splenocyte proliferation and IL-12 production, in order to select the most promising compounds for subsequent in vivo experiments. We have tested several immunosuppressive agents: thiophosphate oligodeoxynucleotides (A151, ODN2088 and ODN4084-F), which include G-rich regions, as well as their analogues, i.e., thiophosphate oligodeoxynucleotides with mesylphosphoramide (ì) modifications at GpG bonds (ì-A151, ì-ODN2088 and ì-ODN4084-F). The effects of chemically modified oligonucleotides were assessed in the in vitro model of CpG-stimulated splenocytes, using CpG-ODN SD-101 in its complete thiophosphate (PS) version. Primary in vitro screening of immunosuppressive oligonucleotides by their effect on splenocyte proliferation and IL-12 production enabled us to identify the most active compounds and determine the features of sequences with the most pronounced immunosuppressive properties, as well as establish optimal concentrations of the studied oligodeoxynucleotides selected for subsequent in vivo studies.

Enzymatic bypass of G-quadruplex structures containing oxidative lesions.

The function of many DNA processing enzymes involves sliding along the double helix or individual DNA strands. Stable secondary structures in the form of G-quadruplexes are difficult for such enzymes to bypass. We used a polymerase stop assay to determine which structural features of the human telomeric and the BCL2 promoter G-quadruplexes can stall progression of the Klenow fragment. Primer extension profiles revealed that G-quartets are effective roadblocks for the Klenow fragment, while auxiliary base pairs can be easily bypassed. Furthermore, we utilized 8-oxoguanine to simulate oxidative damage in G-rich regions and determine the effects on enzyme bypass. In rare cases, oxidative lesions reduce the level of G-quadruplex bypass. In general, however, oxidative lesions reduce G-quadruplex stability and facilitate bypassing of such G-rich regions, especially if the lesion persists in unfolding intermediates. Our findings using Klenow fragment can be extrapolated to other G-quadruplex forming sequences and enzymes that utilise a clamp-like structure to slide along DNA and are involved in processes such as gene expression regulation and telomere maintenance.

Widespread 3′UTR capped RNAs derive from G-rich regions in proximity to AGO2 binding sites

The 3′ untranslated region (3′UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis of gene expression (CAGE), a method for the detection of capped 5′ ends of mRNAs, additionally reveals a large number of apparently 5′ capped RNAs derived from locations within the body of the transcript, including 3′UTRs. Here, we provide direct evidence that these 3′UTR-derived RNAs are indeed capped and widespread in mammalian cells. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA treatment, we find that these 3′UTR-derived RNAs likely originate from AGO2-binding sites, and most often occur at locations with G-rich motifs bound by the RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validate several 3′UTR-derived RNAs, showcase their variable abundance and show that they may not co-localise with the parental mRNAs. Taken together, we provide new insights into the origin and prevalence of 3′UTR-derived RNAs, show the utility of CAGE-seq for their genome-wide detection and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs.Graphical Schematic representation of the proposed model where 3′UTR-derived RNAs originate from G-rich regions enriched in AGO2 and UPF1 binding sites.

Decoding the Role of NEIL1 Gene in DNA Repair and Lifespan: A Literature Review with Bioinformatics Analysis.

Longevity, the length of an organism's lifespan, is impacted by environmental factors, metabolic processes, and genetic determinants. The base excision repair (BER) pathway is crucial for maintaining genomic integrity by repairing oxidatively modified base lesions. Nei-like DNA Glycosylase 1 (NEIL1), part of the BER pathway, is vital in repairing oxidative bases in G-rich DNA regions, such as telomeres and promoters. Hence, in this comprehensive review, it have undertaken a meticulous investigation of the intricate association between NEIL1 and longevity. The analysis delves into the multifaceted aspects of the NEIL1 gene, its various RNA transcripts, and the diverse protein isoforms. In addition, a combination of bioinformatic analysis is conducted to identify NEIL1 mutations, transcription factors, and epigenetic modifications, as well as its lncRNA/pseudogene/circRNA-miRNA-mRNA regulatory network. The findings suggest that the normal function of NEIL1 is a significant factor in human health and longevity, with defects in NEIL1 potentially leading to various cancers and related syndromes, Alzheimer's disease, obesity, and diabetes.

Characterization of G-quadruplexes in the Helicobacter pylori genome and assessment of therapeutic potential of G4 ligands.

Helicobacter pylori, a leading human pathogen associated with duodenal ulcer and gastric cancer, presents a significant threat to human health due to increasing antibiotic resistance rates. This study investigates G-quadruplexes (G4s), which are non-canonical secondary structures form in G-rich regions within the H. pylori genome. Extensive research on G4s in eukaryotes has revealed their role in epigenetically regulating cellular processes like gene transcription, DNA replication, and oncogene expression. However, understanding of G4-mediated gene regulation in other organisms, especially bacterial pathogens, remains limited. Although G4 motifs have been extensively studied in a few bacterial species such as Mycobacterium, Streptococci, and Helicobacter, research on G4 motifs in other bacterial species is still sparse. Like in other organisms such as archaea, mammals, and viruses, G4s in H. pylori display a non-random distribution primarily situated within open reading frames of various protein-coding genes. The occurrence of G4s in functional regions of the genome and their conservation across different species indicates that their placement is not random, suggesting an evolutionary pressure to maintain these sequences at specific genomic sites. Moreover, G-quadruplexes show enrichment in specific gene classes, suggesting their potential involvement in regulating the expression of genes related to cell wall/membrane/envelope biogenesis, amino acid transport, and metabolism. This indicates a probable regulatory role for G4s in controlling the expression of genes essential for H. pylori survival and virulence. Biophysical techniques such as Circular Dichroism spectroscopy and Nuclear Magnetic Resonance were used to characterize G4 motifs within selected H. pylori genes. The study revealed that G-quadruplex ligand inhibited the growth of H. pylori, with minimal inhibitory concentrations in the low micromolar range. This suggests that targeting G4 structures could offer a promising approach for developing novel anti-H. pylori drugs.

Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol β, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.

Unique characteristics of the J-domain proximal regions of Hsp70 cochaperone Apj1 in prion propagation/elimination and its overlap with Sis1 function.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s. The Class A JDP Apj1 of the yeast cytosol has an unusually complex region between the N-terminal J-domain and the substrate binding region-often called the Grich or GF region in Class A and B JDPs because of its typical abundance of glycine. The N-terminal 161-residue Apj1 fragment is known to be sufficient for Apj1 function in prion curing, driven by the overexpression of Hsp104. Further analyzing the N-terminal segment of Apj1, we found that a 90-residue fragment that includes the 70-residue J-domain and the adjacent 12-residue glutamine/alanine (Q/A) segment is sufficient for curing. Furthermore, the 121-residue fragment that includes the Grich region was sufficient to not only sustain the growth of cells lacking the essential Class B JDP Sis1 but also enabled the maintenance of several prions normally dependent on Sis1 for propagation. A J-domain from another cytosolic JDP could substitute for the Sis1-related functions but not for Apj1 in prion curing. Together, these results separate the functions of JDPs in prion biology and underscore the diverse functionality of multi-domain cytosolic JDPs in yeast.

Abstract CT105: Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug

Abstract The human genome contains G-rich regions that are over-represented in many cancer gene promoters. These can form higher-order G-quadruplex arrangements under the influence of an appropriate and selective small molecule compound. The resulting ligand-G-quadruplex complex may be resistant to polymerase read-through and thus results in transcriptional down-regulation of target genes. QN-302, based on a tetra-substituted naphthalene diimide chemotype, is a potent and selective G-quadruplex-binding compound that has been designed and initially developed at University College London. It displays single-digit cell growth inhibition in a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines. RNA-seq analysis shows that it is a pan-quadruplex agent, down-regulating the expression of multiple G-quadruplex-promoter cancer genes and pathways in PDAC. It also shows in vivo anti-tumor activity in several animal models for PDAC, with low toxicity observed at therapeutic doses. QN-302 was licensed to Qualigen Therapeutics Inc in January 2022. It was granted Orphan Drug designation by the FDA in December 2022 for PDAC. This was followed by extensive regulatory toxicological and pharmacological studies together with successful scale-up synthesis, purification and formulation. QN-302 was granted IND clearance in August 2023 by the FDA for phase 1 clinical trials in advanced or metastatic solid tumors. This Phase 1a clinical trial is being conducted in two USA cancer centers (START Midwest, Grand Rapids, MI and Honor Health, Scottsdale, AZ) using a protocol based on 28-day once-weekly dose escalation and evaluation of three potential biomarkers, two of which have been suggested by transcriptomic analysis as being indicative of G-quadruplex-induced drug response and accompanying decrease of anti-tumor activity. So far 1 Cohort (3 patients) has been dosed at the lowest dose of 1.62 mg of QN-302 as part of this Phase 1a clinical trial. Patient #1, a male patient of metastatic pancreatic cancer, is currently in Cycle 4 of therapy showing Stable Disease and no AEs or SAEs reported. Patient #2, a female patient of metastatic colorectal cancer, completed Cycle 2 after which she showed progression upon CT Scan before entering Cycle 3 and was taken off study. Patient #3, a male patient of metastatic PDAC, is currently ending Cycle 2, about to enter Cycle 3, showing Stable Disease upon CT scan. All patients dosed showed no AEs, SAEs or side effects. They were also clinically observed to have improved QOL compared to prior SOC while on therapy with QN-302, This presentation will outline this unique approach and summarize safety, tolerability, PK/PD data and any early clinical data as described above including preliminary biomarker data now that clinical evaluation has commenced. The advantages and challenges of QN-302 will also be discussed. Citation Format: Tariq Arshad, Stephen Neidle, Sreenivasa Chandana, Erkut Borazanci. Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT105.

Abstract 329: NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology

Abstract Background: Telomeres are specialized nucleoprotein structures located at the end of linear chromosomes in eukaryotic cells, and protect the ends of chromosomal DNA from degradation, end-to-end fusion and abnormal recombination. Changes in telomere length have become an important biomarker, which is closely related to cellular aging, all-cause mortality and a variety of diseases. However, there are still challenges in quantifying telomere absolute length, due to repeats of nucleotide sequence (5’-TTAGGG-3’)n in single-stranded G-rich telomeric region, and lack of a method for nucleotide separation and enrichment of individual telomeres. Methods: We developed NanoTelo, a workflow for detecting telomere absolute length based on nanopore sequencing technology. For experimental steps, a self-designed telomere adapter was first connected to the C-rich strand at the end of the chromosome by utilizing the 3' end overhang. And the 5' end of the telomere adapter was complementary to the sequencing adapter, allowing the antisense telomere chain to perform single-strand extension sequencing in the 5' to 3' direction, thus into the sub-telomeric region from the telomere end. The sequencing library was nanopore-sequenced on the QPursue-6k according to the manufacturer’s instructions. For data analysis, reads were strictly extracted (dataset B) with the telomere adapter and library structure in raw sequencing data (dataset A). The reads of dataset A and dataset B were corrected, aligned to the human reference genome (T2T-CHM13v2.0), and calculated the telomere length distribution of each chromosomal (autosomes and sex chromosomes) p/q-arm, respectively. Defined the mean telomere length of all chromosomes represented the overall telomere length, the estimated results of dataset A and dataset B were weighted to obtain the final telomere absolute length. Results: We selected 5 cell lines, which was HCT116, 293T, T24, IMR90 and WI38, and performed 2 experimental replicates for each one. We found that the estimated telomere lengths for each cell line were 3,528/3,512 bp, 4,298/4,311 bp, 2,745/2,685 bp, 4,926/4,915 bp and 4,143/4,096 bp, respectively. A comparative analysis with results from the Pacbio sequencing platform for each cell line in the Tham et al.'s paper, showed that there was no significant statistical difference (two-tailed T-test: p-value=0.98), and the mean absolute difference for estimated telomere length between the two platforms was 7.7 bp (95% CI: 5.4-10.0 bp). Conclusions: NanoTelo demonstrated the possibility of measuring telomere absolute length with a single chromosomal p/q-arm at nucleotide-level resolution. We hope that NanoTelo can not only be used as an important tool for estimating telomere length to dynamically monitor aging, but also provide strong technical support for scientific research and/or clinical application related to human anti-aging and disease intervention. Citation Format: Jidong Lang, Yanmei Chen, Zhi Yang, Beibei Huo. NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 329.

Hmo1 Promotes Efficient Transcription Elongation by RNA Polymerase I in Saccharomyces cerevisiae.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

Structural effects of inosine substitution in telomeric DNA quadruplex.

The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, has been shown to adopt higher-order structures, specifically G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition through chromosome maintenance and manipulation of oncogene expression featuring their G-rich promoter regions. Besides higher order structures, several regulatory roles are attributed to DNA epigenetic markers. In this work, we investigated how the structural dynamics of a G-quadruplex, formed by the telomeric sequence, is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG)n telomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG)4, (IGG)4, (GIG)4, (GGI)4, (IGI)4, (IIG)4, (GII)4, and (III)4, bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na+ and K+ containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG)4 and the terminal modified DNAs (IGG)4 and (GGI)4 in both Na+ and K+ containing buffers. Specifically, quadruplex hybrid was observed for (GGG)4 in K+ buffer. Among the other modified sequences, (GIG)4, (IGI)4 and (GII)4 show parallel features, while (IIG)4 and (III)4 show no detectable conformation in the presence of either Na+ or K+. Our studies indicate that terminal lesions (IGG)4 and (GGI)4 may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI)4 in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K+ cation confers more thermodynamic stability compared to Na+. Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with position dependent G-to-I mutations in different ion conditions.

Structural elucidation of HIV-1 G-quadruplexes in a cellular environment and their ligand binding using responsive 19F-labeled nucleoside probes.

Understanding the structure and recognition of highly conserved regulatory segments of the integrated viral DNA genome that forms unique topologies can greatly aid in devising novel therapeutic strategies to counter chronic infections. In this study, we configured a probe system using highly environment-sensitive nucleoside analogs, 5-fluoro-2'-deoxyuridine (FdU) and 5-fluorobenzofuran-2'-deoxyuridine (FBFdU), to investigate the structural polymorphism of HIV-1 long terminal repeat (LTR) G-quadruplexes (GQs) by fluorescence and 19F NMR. FdU and FBFdU, serving as hairpin and GQ sensors, produced distinct spectral signatures for different GQ topologies adopted by LTR G-rich oligonucleotides. Importantly, systematic 19F NMR analysis in Xenopus laevis oocytes gave unprecedented information on the structure adopted by the LTR G-rich region in the cellular environment. The results indicate that it forms a unique GQ-hairpin hybrid architecture, a potent hotspot for selective targeting. Furthermore, structural models generated using MD simulations provided insights on how the probe system senses different GQs. Using the responsiveness of the probes and Taq DNA polymerase stop assay, we monitored GQ- and hairpin-specific ligand interactions and their synergistic inhibitory effect on the replication process. Our findings suggest that targeting GQ and hairpin motifs simultaneously using bimodal ligands could be a new strategy to selectively block the viral replication.

Simple, sensitive, and label-free miRNA analysis through strand displacement reaction integrating with G-quadruplex-based signal generation

AbstractMicroRNAs (miRNAs) play a significant role in the pathogenesis of various diseases throughout biological processes, and the accurate detection of miRNA biomarkers holds great potential for early stage disease diagnosis and treatment. In this study, a novel method is developed to detect miRNA-21, a biomarker for drug-induced liver injury, by combining target sequence recycling with G-quadruplex-based signal production. This approach is highly sensitive and does not require the use of labels. The target sequence facilitates the cyclic exposure of G-rich regions in the detection probe by toehold-mediated strand displacement processes, with the aid of the catalytic chain. The G-quadruplex sequences that have been produced subsequently interact with thioflavin T (ThT), resulting in a significant increase in its fluorescence intensity. This enhanced fluorescence is utilized for the purpose of detecting miRNA-21, with a remarkably low detection limit of 4.4 fM. The suggested technique also allows for the very specific identification of the target miRNA-21. Due to its non-label format, excellent selectivity, and sensitivity, this technology presents a straightforward and versatile approach for detecting a wide range of biomarkers in the early phases of illness detection.

Comparative analysis of the myoglobin gene in whales and humans reveals evolutionary changes in regulatory elements and expression levels.

Cetacea and other diving mammals have undergone numerous adaptations to their aquatic environment, among them high levels of the oxygen-carrying intracellular hemoprotein myoglobin in skeletal muscles. Hypotheses regarding the mechanisms leading to these high myoglobin levels often invoke the induction of gene expression by exercise, hypoxia, and other physiological gene regulatory pathways. Here we explore an alternative hypothesis: that cetacean myoglobin genes have evolved high levels of transcription driven by the intrinsic developmental mechanisms that drive muscle cell differentiation. We have used luciferase assays in differentiated C2C12 cells to test this hypothesis. Contrary to our hypothesis, we find that the myoglobin gene from the minke whale, Balaenoptera acutorostrata, shows a low level of expression, only about 8% that of humans. This low expression level is broadly shared among cetaceans and artiodactylans. Previous work on regulation of the human gene has identified a core muscle-specific enhancer comprised of two regions, the "AT element" and a C-rich sequence 5' of the AT element termed the "CCAC-box". Analysis of the minke whale gene supports the importance of the AT element, but the minke whale CCAC-box ortholog has little effect. Instead, critical positive input has been identified in a G-rich region 3' of the AT element. Also, a conserved E-box in exon 1 positively affects expression, despite having been assigned a repressive role in the human gene. Last, a novel region 5' of the core enhancer has been identified, which we hypothesize may function as a boundary element. These results illustrate regulatory flexibility during evolution. We discuss the possibility that low transcription levels are actually beneficial, and that evolution of the myoglobin protein toward enhanced stability is a critical factor in the accumulation of high myoglobin levels in adult cetacean muscle tissue.

Conversion to Trimolecular G-Quadruplex by Spontaneous Hoogsteen Pairing-Based Strand Displacement Reaction between Bimolecular G-Quadruplex and Double G-Rich Probes.

Bimolecular or tetramolecular G-quadruplexes (GQs) are predominantly self-assembled by the same sequence-identical G-rich oligonucleotides and usually remain inert to the strand displacement reaction (SDR) with other short G-rich invading fragments of DNA or RNA. Appealingly, in this study, we demonstrate that a parallel homomeric bimolecular GQ target of Tub10 d(CAGGGAGGGT) as the starting reactant, although completely folded in K+ solution and sufficiently stable (melting temperature of 57.7 °C), can still spontaneously accept strand invasion by a pair of short G-rich invading probes of P1 d(TGGGA) near room temperature. The final SDR product is a novel parallel heteromeric trimolecular GQ (tri-GQ) of Tub10/2P1 reassembled between one Tub10 strand and two P1 strands. Here we present, to the best of our knowledge, the first NMR solution structure of such a discrete heteromeric tri-GQ and unveil a unique mode of two probes vs one target in mutual recognition among G-rich canonical DNA oligomers. As a model system, the short invading probe P1 can spontaneously trap G-rich target Tub10 from a Watson-Crick duplex completely hybridized between Tub10 and its fully complementary strand d(ACCCTCCCTG). The Tub10 sequence of d(CAGGGAGGGT) is a fragment from the G-rich promoter region of the human β2-tubulin gene. Our findings provide new insights into the Hoogsteen pairing-based SDR between a GQ target and double invading probes of short G-rich DNA fragments and are expected to grant access to increasingly complex architectures in GQ-based DNA nanotechnology.

Crystal structure of a cap-independent translation enhancer RNA.

In eukaryotic messengerRNAs, the 5' cap structure binds to the translation initiation factor 4E to facilitate early stages of translation. Although many plant viruses lack the 5' cap structure, some contain cap-independent translation elements (CITEs) in their 3' untranslated region. The PTE (Panicum mosaic virus translation element) class of CITEs contains a G-rich asymmetric bulge and a C-rich helical junction that were proposed to interact via formation of a pseudoknot. SHAPE analysis of PTE homologs reveals a highly reactive guanosine residue within the G-rich region proposed to mediate eukaryotic initiation factor 4E (eIF4E) recognition. Here we have obtained the crystal structure of the PTE from Pea enation mosaic virus 2 (PEMV2) RNA in complex with our structural chaperone, Fab BL3-6. The structure reveals that the G-rich and C-rich regions interact through a complex network of interactions distinct from those expected for a pseudoknot. The motif, which contains a short parallel duplex, provides a structural mechanism for how the guanosine is extruded from the core stack to enable eIF4E recognition. Homologous PTE elements harbor a G-rich bulge and a three-way junction and exhibit covariation at crucial positions, suggesting that the PEMV2 tertiary architecture is conserved among these homologs.

Cooperative interaction of CST and RECQ4 resolves G-quadruplexes and maintains telomere stability.

Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that interacts with the replisome to aid in stalled fork rescue. We previously found that CST promotes telomere replication to maintain genomic integrity via G-quadruplex (G4) resolution. However, the detailed mechanism by which CST resolves G4s in vivo and whether additional factors are involved remains unclear. Here, we identify RECQ4 as a novel CST-interacting partner and show that RECQ4 can unwind G4 structures in vitro using a FRET assay. Moreover, G4s accumulate at the telomere after RECQ4 depletion, resulting in telomere dysfunction, including the formation of MTSs, SFEs, and TIFs, suggesting that RECQ4 is crucial for telomere integrity. Furthermore, CST is also required for RECQ4 telomere or chromatin localization in response to G4 stabilizers. RECQ4 is involved in preserving genomic stability by CST and RECQ4 disruption impairs restart of replication forks stalled by G4s. Overall, our findings highlight the essential roles of CST and RECQ4 in resolving G-rich regions, where they collaborate to resolve G4-induced replication deficiencies and maintain genomic homeostasis.

Helquat dyes targeting G-quadruplexes as a new class of anti-HIV-1 inhibitors

The secondary structure of nucleic acids containing quartets of guanines, termed G-quadruplexes, is known to regulate the transcription of many genes. Several G-quadruplexes can be formed in the HIV-1 long terminal repeat promoter region and their stabilization results in the inhibition of HIV-1 replication. Here, we identified helquat-based compounds as a new class of anti-HIV-1 inhibitors that inhibit HIV-1 replication at the stage of reverse transcription and provirus expression. Using Taq polymerase stop and FRET melting assays, we have demonstrated their ability to stabilize G-quadruplexes in the HIV-1 long-terminal repeat sequence. Moreover, these compounds were not binding to the general G-rich region, but rather to G-quadruplex-forming regions. Finally, docking and molecular dynamics calculations indicate that the structure of the helquat core greatly affects the binding mode to the individual G-quadruplexes. Our findings can provide useful information for the further rational design of inhibitors targeting G-quadruplexes in HIV-1.

Taq-Polymerase Stop Assay to Determine Target Selectivity of G4 Ligands in Native Promoter Sequences of MYC, TERT, and KIT Oncogenes.

Computational and high-throughput experimental methods predict thousands of potential quadruplex sequences (PQSs) in the human genome. Often these PQSs contain more than four G-runs, which introduce additional uncertainty into the conformational polymorphism of the G4 DNA. G4-specific ligands, which are currently being actively developed as potential anticancer agents or tools for studying G4 structures in genomes, may preferentially bind to specific G4 structures over the others that can be potentially formed in the extended G-rich genomic region. We propose a simple technique that identifies the sequences that tend to form G4 in the presence of potassium ions or a specific ligand. Thermostable DNA Taq-polymerase stop assay can detect the preferential position of the G4 -ligand binging within a long PQS-rich genomic DNA fragment. This technique was tested for four G4 binders PDS, PhenDC3, Braco-19, and TMPyP4 at three promoter sequences of MYC, KIT, and TERT that contain several PQSs each. We demonstrate that the intensity of polymerase pausing reveals the preferential binding of a ligand to particular G4 structures within the promoter. However, the strength of the polymerase stop at a specific site does not always correlate with the ligand-induced thermodynamic stabilization of the corresponding G4 structure.

Protein-RNA interactions of FMRP and CCND1: Implications of regulation in neurogenesis.

While originally discovered in the 1960s, adult neurogenesis, the production of mature neurons from neuronal stem cells in adults, has become largely accepted by the field only in the 1990s. Thus, the detailed mechanisms of its regulation remain elusive, requiring further characterization of the protein players involved in this process. Cyclin-D1, a regulatory subunit of cyclin-dependent kinases involved in monitoring entry into the cell cycle, has been identified as a key factor in adult neurogenesis and is encoded by the CCND1 gene. Another identified component of this process is the Fragile X messenger ribonucleoprotein (FMRP), an RNA-binding protein associated with translational regulation. Interestingly, FMRP has been shown to regulate cyclin-D1 expression, emphasizing a potential interplay between FMRP function and cyclin-D1 expression in neurons. FMRP has shown high affinity binding for guanine quadruplex (GQ) forming RNAs. We identified that CCND1 mRNA has a G-rich region in its 3’-untranslated region that has the potential to form GQ structures, and thus possibly interacting with FMRP through the recognition of these structures. To elucidate the presence of a GQ in the CCND1 mRNA, we used biophysical methods, such as 1H NMR, UV thermal denaturation, CD spectroscopy, and native polyacrylamide gel electrophoresis (PAGE). This study effectively characterizes a GQ in the 3’UTR of the CCND1 mRNA and its interactions with FMRP, providing insight on additional regulatory mechanisms of adult neurogenesis and guiding development of novel therapeutic methods.