Guanine Residues Research Articles

The influence of G-tract and loop length on the topological variability of putative five and six G-quartet DNA structures in the human genome

Local variation of DNA structure and its dynamic nature play an essential role in the regulation of important biological processes. One of the most prominent noncanonical structures are G-quadruplexes, which form in vivo within guanine-rich regions and have been demonstrated to be involved in the regulation of transcription, translation and telomere maintenance. We provide an analysis of G-quadruplex formation in sequences with five and six guanine residues long G-tracts, which have emerged from the investigation of the gapless human genome and are associated with genes related to cancer and neurodegenerative diseases. We systematically explored the effect of G-tract and loop elongations by means of NMR and CD spectroscopy and polyacrylamide electrophoresis. Despite both types of elongation leading up to structural polymorphism, we successfully determined the topologies of four out of eight examined sequences, one of which contributes to a very scarce selection of currently known intramolecular four G-quartet structures in potassium solutions. We demonstrate that examined sequences are incompatible with five or six G-quartet structures with propeller loops, although the compatibility with other loop types cannot be factored out. Lastly, we propose a novel approach towards specific G-quadruplex targeting that could be implemented in structures with more than four G-quartets.

Abstract A014: Protein-informed gene methylation signatures to predict treatment response and outcomes in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer with dismal outcomes. We need new biomarkers to optimize the limited treatment combinations used in current clinical practice. Many machine-learning models have been presented based on mutational or transcriptome data claiming to predict outcomes. Still, they do not provide actionable targets (genes) that could improve treatment response. Moreover, DNA-methylation changes that influence gene/protein expression have been explored for this purpose. From the literature, we curated a 122-gene panel (January 1970 to April 2024) constructed on the proteins with preclinical (on cell lines or mouse models) or clinical (tissue-based studies) evidence suggesting their individual roles in influencing response to drugs often used in managing PDA such as gemcitabine (Gem), nab-paclitaxel, oxaliplatin, 5-fluorouracil, irinotecan, and cisplatin. We hypothesized that methylation changes in these genes are surrogates for gene/protein expression and tested their clinical significance in The Cancer Genome Atlas (TCGA) PDA population (n=184). The goal was to develop methylation signatures composed of specific cytosines followed by guanine residue (CpG) sites that aid in identifying patients at high risk of treatment failure and poor outcomes. We used elastic net multivariate analysis to calculate survival and plot Kaplan Meier plots based on our gene panel. The alpha (α) used for this script was calculated based on the lowest mean error and the minimum lambda (λ) value accordingly for each iteration (100 iterations to calculate the best values for α and λ). Our analysis yielded 28 methylation signatures that could identify high-risk PDA patients with poor overall survival (OS). The number of CpG sites in these signatures ranged from 1 to 1933. The differences in OS for these signatures were better than those available in the literature. The best signature had 18 CpG sites (17 months (m) vs. not evaluable) followed by a 53-CpG site signature (16 m vs. 67m) and a 6-CpG site signature (17m vs. 67m). A signature with the highest CpG sites (1933) was not better than those with smaller ones (16m vs. 21m). Multiple signatures had >1 CpG sites from one gene. Early growth response 1 (EGR1) coding the KCNH2 gene has featured in most signatures. One CpG site in this gene had a clinically significant prognostic value (15m vs. 30m). Other frequently featured genes were CCDC85A, EGFR, SST, and HMGA1. The main drawbacks for this study were that Gem-related genes (> 60) dominated the panel, and those with Gem treatment dominated the patient population. This panel should be further refined to make it more relevant to current clinical practice. We provide preliminary evidence suggesting the clinical significance of our panel and lay the foundation for future studies that would enable us to personalize prognostication and treatment selection in PDA patients. Our work can provide novel therapeutic targets that, alone or in combination with chemotherapy, can improve the outcomes for PDA. Citation Format: Ashish Manne, Harshitha Puram, Deepak Sherpally, Sravan Jeepalyam, Upender Manne. Protein-informed gene methylation signatures to predict treatment response and outcomes in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A014.

DNA Repair and Mutagenesis of ADP-Ribosylated DNA by Pierisin.

Pierisin is a DNA-targeting ADP-ribosyltransferase found in cabbage white butterfly (Pieris rapae). Pierisin transfers an ADP-ribosyl moiety to the 2-amino group of the guanine residue in DNA, yielding N2-(ADP-ribos-1-yl)-2'-deoxyguanosine (N2-ADPR-dG). Generally, such chemically modified DNA is recognized as DNA damage and elicits cellular responses, including DNA repair pathways. In Escherichia coli and human cells, it has been experimentally demonstrated that N2-ADPR-dG is a substrate of the nucleotide excision repair system. Although DNA repair machineries can remove most lesions, some unrepaired damages frequently lead to mutagenesis through DNA replication. Replication past the damaged DNA template is called translesion DNA synthesis (TLS). In vitro primer extension experiments have shown that eukaryotic DNA polymerase κ is involved in TLS across N2-ADPR-dG. In many cases, TLS is error-prone and thus a mutagenic process. Indeed, the induction of G:C to T:A and G:C to C:G mutations by N2-ADPR-dG in the hypoxanthine phosphoribosyltransferase gene mutation assay with Chinese hamster cells and supF shuttle vector plasmids assay using human fibroblasts has been reported. This review provides a detailed overview of DNA repair, TLS and mutagenesis of N2-ADPR-dG induced by cabbage butterfly pierisin-1.

Interactions with DNA Models of the Oxaliplatin Analog (cis-1,3-DACH)PtCl2.

It is generally accepted that adjacent guanine residues in DNA are the primary target for platinum antitumor drugs and that differences in the conformations of the Pt-DNA adducts can play a role in their antitumor activity. In this study, we investigated the effect of the carrier ligand cis-1,3-diaminocyclohexane (cis-1,3-DACH) upon formation, stability, and stereochemistry of the (cis-1,3-DACH)PtG2 and (cis-1,3-DACH)Pt(d(GpG)) adducts (G = 9-EthlyGuanine, guanosine, 5'- and 3'-guanosine monophosphate; d(GpG) = deoxyguanosil(3'-5')deoxyguanosine). A peculiar feature of the cis-1,3-DACH carrier ligand is the steric bulk of the diamine, which is asymmetric with respect to the Pt-coordination plane. The (cis-1,3-DACH)Pt(5'GMP)2 and (cis-1,3-DACH)Pt(3'GMP)2 adducts show preference for the ΛHT and ∆HT conformations, respectively (HT stands for Head-to-Tail). Moreover, the increased intensity of the circular dichroism signals in the cis-1,3-DACH derivatives with respect to the analogous cis-(NH3)2 species could be a consequence of the greater bite angle of the cis-1,3-DACH carrier ligand with respect to cis-(NH3)2. Finally, the (cis-1,3-DACH)Pt(d(GpG)) adduct is present in two isomeric forms, each one giving a pair of H8 resonances linked by a NOE cross peak. The two isomers were formed in comparable amounts and had a dominance of the HH conformer but with some contribution of the ΔHT conformer which is related to the HH conformer by having the 3'-G base flipped with respect to the 5'-G residue.

Small molecule telomerase inhibitors are also potent inhibitors of telomeric C-strand synthesis.

Telomere replication is essential for continued proliferation of human cells, such as stem cells and cancer cells. Telomerase lengthens the telomeric G-strand, while C-strand replication is accomplished by CST-polymerase α-primase (CST-PP). Replication of both strands is inhibited by formation of G-quadruplex (GQ) structures in the G-rich single-stranded DNA. TMPyP4 and pyridostatin (PDS), which stabilize GQ structures in both DNA and RNA, inhibit telomerase in vitro, and in human cells they cause telomere shortening that has been attributed to telomerase inhibition. Here, we show that TMPyP4 and PDS also inhibit C-strand synthesis by stabilizing DNA secondary structures and thereby preventing CST-PP from binding to telomeric DNA. We also show that these small molecules inhibit CST-PP binding to a DNA sequence containing no consecutive guanine residues, which is unlikely to form GQs. Thus, while these "telomerase inhibitors" indeed inhibit telomerase, they are also robust inhibitors of telomeric C-strand synthesis. Furthermore, given their binding to GQ RNA and their limited specificity for GQ structures, they may disrupt many other protein-nucleic acid interactions in human cells.

Evaluation of VAL-083 in GBM AGILE, a phase 3 registration platform trial for newly diagnosed and recurrent glioblastoma.

2005 Background: GBM AGILE (NCT03970447) is a phase 2/3 Bayesian adaptive registration platform trial testing multiple therapies efficiently against a common control (C) with a primary endpoint of overall survival (OS). VAL-083 (VAL) is a DNA targeting agent that, independent of O6-methylguanine DNA methyltransferase promoter methylation status, targets the N7 position of guanine residues and facilitates inter-strand DNA crosslinks, leading to DNA double-strand breaks and cell death. It entered the trial in January 2021, and it is the 2nd arm (of 6) to complete its evaluation. Methods: Patient subtypes considered in GBM AGILE are newly diagnosed methylated (NDM), ND unmethylated (NDU), & recurrent disease (RD). C is temozolomide (in ND) & lomustine (in RD). Arms open to all 3 subtypes are evaluated in 5 prospectively defined signatures (sig): NDU, NDM, RD, all ND and All. Randomization to C is 20% in each subtype. Exp arms in GBM AGILE have 1 or 2 stages. Efficacy is based on OS hazard ratio (HR) of arm/C. Efficacy goal is a final Bayesian probability ≥ 98% for HR <1.00 in combined Stages 1 & 2. Arms stop accruing in Stage 1 if they reach max sample size (N) or drop for futility or safety. Exp arms in Stage 1 are adaptively randomized with allocation being proportional to an arm’s current probability of having ≥ 30% benefit in OS, P(HR <0.70). In stage 1, exp arms are evaluated monthly, and arms showing Bayesian predictive power (PP) ≥ 0.8, graduate into Stage 2 with fixed randomization in one sig. For all exp arms, follow up continues for 12 mos after accrual stops (clinical cutoff). Arms are declared futile at any monthly analysis when PP is <0.25 for all sigs. Open to all 3 subtypes, VAL entered as the 1st arm in NDM and was randomized 1:1 to C in this subtype until additional arms entered. The target max N for VAL in its Stages 1 & 2 were 150 and 50, resp. Results: At the interim after VAL reached max sample size in Stage 1, the PP for all signatures was <0.8 and >0.25 for at least one sig. Thus, VAL did not graduate nor drop for futility, but accrual stopped for maximum N in Stage 1 (see table). Final results will be presented at the meeting. Columns 2-5 show results at the interim after which VAL stopped for max N. Columns 6-8 show near final results. Conclusions: GBM AGILE is an efficient & effective model for phase 3 drug development. VAL did not increase OS compared to C in any glioblastoma subtype. GBM AGILE evaluated this agent in less time, at lower cost, & with fewer patients than typical registration trials & is currently evaluating several other arms. Clinical trial information: NCT03970447 . [Table: see text]

Abstract 3814: Molecular background conditioning by temozolomide in neuroendocrine tumors

Abstract Temozolomide (TMZ)-based regimens represent a valid therapeutic option in well-differentiated neuroendocrine tumors (NETs G2 and G3). As an alkylating agent, it increases DNA alteration by altering guanine residues, which either triggers cell death or results in novel missense mutations especially in MGMT deficient cells. Here, we describe an exploratory case series of TMZ-treated NETs cases that underwent NGS analysis at the University Hospital of Verona. Methods. We included 32 patients (16 G2 and 16 G3). NGS was performed before TMZ treatment for 22 cases (54%), after for 19 (46%) and in both for 4 cases (13%). We performed NGS on tissue samples for 73% of assays and via liquid biopsy for the remaining 27%. For tissue NGS, in 77% of samples we used an in-house panel encompassing 174 genes and in 23% commercial test interrogating at least 500 genes. Only ACMG/AMP class IV-V molecular alterations were considered for further analysis. Main Results From 2021 we performed NGS analysis on 32 NETs (G2 and G3) treated with TMZ-based regimes. Most cases (28 pf 32, 84.4%) were pancreatic NETs. Mean Ki-67 at baseline was 24.5% (range 3-60). The mean number treatments prior to TMZ were 1.4 and 0.5 between CAPTEM and subsequent NGS. The top five altered genes at baseline were MEN1 (32%), TP53 (14%), DAXX (14%), PTEN (9%) and SMAD4 (9%). None of the patients showed a high TMB (>15Mut/Mb) at baseline, while 26% of post-TMZ samples displayed that feature. Mean TMB at baseline and after treatment was 6,51 (range 1-10.6) and 22,9 mut/Mb (range 1-216) respectively. Alterations in DDR systems genes, including ATM, ATR, CHEK2, BRCA1, BRCA2, MLH1, MLH2, PMS2 MLH6 and MUTYH, were found in 8 patients (47%) after TMZ exposure; only one DDR-specific alteration (MUTYH) was found at baseline. DDR defects were enriched in patients exposed to higher numbers of treatments. The increase in mean TMB and enrichment in mutations in the DDR system is also confirmed when analysing the 6 cases having both pre- and post-TMZ treatment samples. Among other pathways, cell cycle deregulating genes (CDKN1A, CDKN2B, CDKN1B, CDK12, CDK6, RB1) were altered in 2 (11%) and 5 (29%) pre- and post-TMZ NGS samples respectively, the mTOR pathway (MTOR, NF1, PIK3CA, AKT, TSC1, TSC2, PTEN) in 2 (11%) and 8 (47%), and the MAPK pathway (KRAS and BRAF) in 1 (5%) and 2 (12%). Conclusions. The wide and effective use of temozolomide in neuroendocrine tumors, together with the potential therapeutic implications of DDR defects make this topic of great interest. The DDR pathway was one of the most affected by alterations in post-TMZ samples, although the genotoxic agent caused a general increase in the rate of somatic mutation. The status of DDR systems, either germinal or somatic, may represent an important selection and/or stratification parameter for future immunotherapy clinical studies also in neuroendocrine tumours provided that the analysis is carried out in a systematic, comprehensive and longitudinal way. Citation Format: Elena Trevisani, Anna Reni, Irene Torresan, Alice Rossi, Michele Borghesani, Silvia Nicolini, Claudio Luchini, Luca Landoni, Davide Melisi, Aldo Scarpa, Michele Milella, Andrea Mafficini, Sara Cingarlini. Molecular background conditioning by temozolomide in neuroendocrine tumors [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 3814.

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation

Key messageBarley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5′ terminal nucleotide of small RNAs, demonstrating functional conservation and divergence.The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5′ adenine residue, while also accepting 5′ guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5′ adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

Identification of neuropathogenic Varicellovirus equidalpha1 as a potential cause of respiratory disease outbreaks among horses in North Xinjiang, China, from 2021-2023.

Varicellovirus equidalpha1 (formerly Equid alphaherpesvirus 1, EqAHV-1) is among the most important viruses responsible for respiratory disease outbreaks among horses throughout the world. No reports to date have detailed the association between EqAHV-1 and respiratory disease among horses in China. This study described one such outbreak among a population of horses in north Xinjiang that occurred from April 2021 - May 2023. qPCR revealed that EqAHV-1 was detectable in all samples and this virus was identified as a possible source of respiratory disease, although a limited subset of these samples were also positive for EqAHV-2, EqAHV-4, and EqAHV-5. In total, three EqAHV-1 strains responsible for causing respiratory illness in horses were isolated successfully, and full-length ORF33 sequence comparisonsand phylogenetic analyses indicated that these isolates may have originated from EqAHV-1 strains detected in Yili horse abortions. ORF30 sequence data additionally suggested that these strains were neuropathic, as evidenced by the presence of a guanine residue at nucleotide position 2254 corresponding to the aspartic acid present at position 752 in the DNA polymerase encoded by this virus. This study is the first report of an outbreak of respiratory disease among horses in China caused by EqAHV-1. ORF30 sequence characterization revealed that these EqAHV-1 strains harbored a neuropathogenic genotype. Given the detection of this virus in horses suffering from respiratory disease, concern is warranted with respect to this neuropathogenic EqAHV-1 outbreak.

High-Resolution Structure of RNA G-Quadruplex Containing Unique Structural Motifs Originating from the 5'-UTR of Human Tyrosine Kinase 2 (TYK2).

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor-associated tyrosine kinases together with highly homologous JAK1, JAK2, and JAK3 paralogues. Overexpression of TYK2 is associated with several inflammatory diseases, including severe complications during the COVID-19 infection. Since the downregulation of JAK paralogues could lead to serious health consequences or even death, it is critical to avoid it when designing drugs to suppress TYK2. To achieve the required specificity only for TYK2, researchers have recently selectively targeted TYK2 mRNA by developing antisense oligonucleotides. In this work, we expand the target space of TYK2 mRNA by showing that the mRNA adopts tetra-helical noncanonical structures called G-quadruplexes. We identified a TYKwt RNA oligonucleotide from the 5'-UTR of TYK2 mRNA, which adopts multiple different parallel G-quadruplexes that exist at equilibrium. Using NMR spectroscopy, we showed that some of the G-quadruplexes adopt unique structural motifs, mainly due to the formation of a stable GA bulge. Using guanine to uridine substitutions, we prepared the oligonucleotide TYK3_U6, which serves as an excellent model for the bulged G-quadruplexes formed by the TYKwt oligonucleotide. NMR structural analysis, including data on the residual coupling constants (RDC) of the loop regions, unveiled that the studied three-quartet parallel G-quadruplex contains many unusual structural features such as a G(U)A bulge, a guanine residue in the syn conformation, A and U residues stacked on the top G-quartet, and a well-defined adenine from a three-residue long propeller loop oriented in the groove, all of which could be valuable targets for future drug design.

Electrochemical and theoretical studies of the interaction between anticancer drug ponatinib and dsDNA

In this study, electrochemical and theoretical studies were performed to explain the interaction mechanism between ponatinib (PNT), a third generation tyrosine kinase inhibitor, and dsDNA. The electrochemical part was conducted in phosphate-buffered saline (PBS) at physiological pH of 7.4 and in acetate buffer with a pH of 4.7, using square wave voltammetry. A boron-doped diamond electrode was used in a bulk-incubated solution. The theoretical part was investigated using computational methods, such as the semiempirical method PM7 and density functional theory (DFT). Significant differences in the electrochemical behavior of PNT in the presence of DNA confirmed the occurrence of interactions. The results obtained in the acetate buffer strongly suggested the preferential interaction of PNT with guanine residues. However, at physiological pH, it can be concluded that PNT interacts with dGua and dAdo in the dsDNA molecule. These results are consistent with outcomes from the theoretical studies, where quantum-chemical calculations showed that both electrochemically detectable nucleobases form hydrogen bonds with the drug. These bonds appeared to be stronger with guanine than with adenine. According to the computational studies, the dsDNA major groove is the energetically preferred site for the complexation of PNT.

Abstract B111: Developing prognostic signatures (ep-Sigs) through epigenetic predictive markers in pancreatic ductal adenocarcinoma (PDA) – A pilot study based on The Cancer Genome Atlas (TCGA) data

Abstract Current genomic testing (mutation profiling) has not proven beneficial for optimal treatment selection or for monitoring drug resistance, which is critical for neoadjuvant therapy (NAT) in borderline resectable/resectable PDA and first-line therapy for advanced PDA. Failure to administer an effective chemotherapy combination results in early progression of the disease, life-threatening complications, and a decline in the patient’s performance status, rendering them ineligible for further anti-cancer treatment. We hypothesized that our panel of epigenetic markers (DNA-methylation changes) is a surrogate for protein/gene expression and will develop an ep-Sigs that also has predictive value. The genes in this panel have been curated from extant literature and belong to one of two broad categories: one with known prognostic value (PvG) and the other with preclinical or clinical evidence of influencing one of the drugs (DrG) used in PDA management (gemcitabine (gem), 5-fluorouracil (5FU), oxaliplatin (Ox), irinotecan (Iri), nab-paclitaxel (NP), paclitaxel (Tax), and cisplatin (Cis)). Some genes/proteins were critical for more than one drug. To accomplish this, we tested the panel in the TCGA pancreatic adenocarcinoma (PAAD) data with 182 patients. For univariate survival analysis (UVA), we used Cox regression models for each marker. For multivariate survival analysis (MVA), we used penalized Cox regression models (i.e., LASSO, Elastic-Net) for identifying marker signatures. The area under the ROC Curve (AUC) was calculated to assess each signature’s predictive performance. We identified 301 cytosines followed by guanine residue (CpG) sites among the 67 genes (gem=35, 5FU=13, 5FU+gem=10, NP=6, Cis=6, and PvG=23) in the panel with significant (p<0.05) association with overall survival (OS) through UVA. We identified five ep-Sigs through MVA that could predict poor OS (p<0.05), which are a combination of markers from PvG and DrG. The number of markers and relevant DrG in them are as follows, 15 & Gem/Cis; 30 & Gem/5FU/Cis/Tax/Iri; 28 & Gem/5FU/NP/Cis/Tax/Iri; 23 & Gem/5FU; 29 & Gem/5FU/NP. We did a similar analysis for RNA-sequencing data from TCGA. We identified 17 markers and 9 signatures on UVA and MVA, respectively. Interestingly, the expression of 100/105 markers in the panel was significantly (p=4.19e-66) high in PDA compared to normal tissues, indicating this panel has diagnostic value too. We believe this project is the first step for this approach, and this concept needs to be evolved with prospective studies and larger samples. The ep-Sigs with a panel of epigenetic markers with a proven influence on resistance to frequently used drugs could help in developing meaningful models that change the approach to managing PDA, especially for NAT. Citation Format: Ashish Manne, Wancai Yang, Davendra Sohal, Ashwini Esnakula, Lianbo Yu. Developing prognostic signatures (ep-Sigs) through epigenetic predictive markers in pancreatic ductal adenocarcinoma (PDA) – A pilot study based on The Cancer Genome Atlas (TCGA) data [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B111.

The Arabidopsis tDR Ala forms G-quadruplex structures that can be unwound by the DExH1 DEA(D/H)-box RNA helicase.

As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5' extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.

Genome sequence and characterization of a novel Pseudomonas putida phage, MiCath

Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products.

Local and cooperative structural transitions of double-stranded DNA in choline-based deep eutectic solvents

The possibility of using deep eutectic solvents (DESs) as co-solvents for stabilizing and preserving the native structure of DNA provides an attractive opportunity in the field of DNA biotechnology. The rationale of this work is a systematic investigation of the effect of hydrated choline-based DES on the structural stability of a 30-base-pair double-stranded DNA model via a combination of spectroscopic experiments and MD simulations. UV absorption and CD experiments provide evidence of a significant contribution of DESs to the stabilization of the double-stranded canonical (B-form) DNA structure. Multi-wavelength synchrotron UV Resonance Raman (UVRR) measurements indicate that the hydration shell of adenine-thymine pairs is strongly perturbed in the presence of DESs and that the preferential interaction between H-bond sites of guanine residues and DESs is significantly involved in the stabilization of the dsDNA. Finally, MD calculations show that the minor groove of DNA is significantly selective for the choline part of the investigated DESs compared to the major groove. This finding is likely to have a significant impact not only in terms of thermal stability but also in the modulation of ligand-DNA interactions.

Forming a chromium-based interstrand DNA crosslink: Implications for carcinogenicity

The reduction of the carcinogen chromate has been proposed to lead to three Cr(III)-containing DNA lesions: binary adducts (Cr(III) and DNA), interstrand crosslinks, and ternary adducts (Cr(III) linking DNA to a small molecule or protein). Although the structures of binary adducts have recently been elucidated, the structures of interstrand crosslinks and ternary adducts are not known. Analysis of Cr(III) binding to an oligonucleotide duplex containing a 5’-CG site allows elucidation of the structure of an oxide- or hydroxide-bridged binuclear Cr(III) assembly bridging the two strands of DNA. One Cr(III) is directly coordinated by the N-7 atom of a guanine residue, and the complex straddles the helix to form a hydrogen bond between another guanine residue and a Cr(III)-bound aquo ligand. No involvement of the phosphate backbone was observed. The properties and stability of this Cr–O(H)–Cr-bridged complex differ significantly from those reported for Cr-induced interstrand crosslinks, suggesting that interstrand crosslinks resulting from chromate reduction may be organic in nature.

Selective Binding Profiles of Curcumin Derivatives to G-Quadruplex (G4) Structures Found in Human Oncogene Promoters

G-Quadruplex (G4) structures are special significant DNA topologies formed by accumulation of G-tetrads which are planar structures of four guanine residues interacting with hydrogen bonds through Hoogsten edges around monovalent cations such as potassium (K) or sodium (Na). While these special topologies are mostly observed in telomere regions, they might be found over regulatory regions of the genes such as promoter, enhancer etc. In addition, since that various oncogenes carry G4 structures over their promoters, it’s highlighted that G4s have significant role over cancer prognosis through regulation of expression level. To date, binding profiles of curcumin having great antioxidant and anti-inflammatory properties and its derivatives to G4s found in telomere regions and promoter of c-Myc were discovered. As such, to discover selective binding profiles of curcumin derivatives to G4s found in promoters of various oncogenes such as c-Myc, c-KIT, hTERT, RET, VEGF, and PARP1 have quite potential in the drug design for several cancer types. In light of these information, 18 curcumin derivatives from ZINC15 database were docked to related G4 structures. ADME and toxicity properties of all derivatives were analyzed and biological reactivity as well as molecular electrostatic surface potential (MESP) features of totally 4 derivatives (C11, C13, C14, and C15) exhibiting selective binding pattern to certain G4s were analyzed with density functional theory (DFT) method.

Analysis on the in-silico ensemble methods for 3D modelling of ssDNA aptamers

Understanding the 3-D structure of nucleic acid aptamers is important for the rational design of aptamer-based constructs in various applications, including for developing aptasensors. Herein, a simple approach for 3D modelling of ssDNA aptamers through an ensemble of web applications has been described. The procedure utilized 30 aptamers whose 3D XRD or NMR experimental structures are available for validation. As a first step, the primary sequences of ssDNA aptamers were transformed into 2D structures using six widely used web applications: RNA fold, Vector builder, RNA Structure, UNA fold, Centroid fold, and IP Knot. The generated 2D structures were then passed through the RNA composer web application to generate 3D RNA structure, which in turn was converted to 3D DNA structures using various Visual Molecular Dynamics web applications that also include conversion of ribose sugar into deoxyribose sugar backbone and uracil to thiamine. The energy-minimized generated 3D structures were matched well with high accuracy to their experimental counterparts. This study identified that the Guanine residues are crucial in the aptamer 3D structure prediction and in algorithms that generate secondary structures. Further, the GC content (<50%), GC bond percentage (<60%), and G:C ratio (<1.12) act as limiting factors in predicting the 2D structures of aptamers. There were variations in the 2D structure predictions by the web applications, even though all these applications were a combination of the MFE, MEA, and McCaskill functions. Processing these structures through the web applications described above produced best-fit 3D structures with the experimental one, thus offering the present ensemble approach to reliably predict the 3D structure of aptamers for various applications.

Hexahelicene DNA-binding: Minor groove selectivity, semi-intercalation and chiral recognition

In this contribution, we focused on a fundamental study targeting the interaction of water-soluble [6]helicene derivative 1 (1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide) with double-stranded (ds) DNA. A synthetic 30-base pair duplex, plasmid, chromosomal calf thymus and salmon DNA were investigated using electrochemistry, electrophoresis and spectroscopic tools supported by molecular dynamics (MD) and quantum mechanical approaches. Both experimental and theoretical work revealed the minor groove binding of 1 to the dsDNA. Both the positively charged imidazole ring and hydrophobic part of the side chain contributed to the accommodation of 1 into the dsDNA structure. Neither intercalation into the duplex DNA nor the stable binding of 1 to single-stranded DNA were found in topoisomerase relaxation experiments with structural components of 1, i.e. [6]helicene (2) and 1-butyl-3-methylimidazolium bromide (3), nor by theoretical calculations. Finally, the binding of optically pure enantiomers (P)-1 and (M)-1 was studied using circular dichroism spectroscopy, isothermal titration calorimetry and UV Resonance Raman (UVRR) methods. Using MD and quantum mechanical methods, minor groove and semi-intercalation were proposed for compound 1 as the predominant binding modes. From the UVRR findings, we also can conclude that 1 tends to preferentially interact with adenine and guanine residues in the structure of dsDNA.

Voltammetric oxidation behavior of single-stranded DNA on carbon screen printed electrodes: From short oligonucleotides to ultralong amplification products

To deeper understand the electrochemical behavior of nucleic acids, the synthetic DNA oligonucleotides of general formula (ACTG)n, where n = 1–12, as well as 16-mer DNA oligonucleotides varying in the number of guanine (dG; G-n, n = 0–7) or adenine (dA; A-n, n = 0–8) residues, and single-stranded DNA (ssDNA) amplification products were employed to systematically investigate their oxidation in the ‘dissolved’ and ‘adsorbed’ forms on carbon screen printed electrodes by square wave and cycling voltammetry in the potential range of 0.5–1.5 V (vs. Ag/AgCl). Two signals at 0.9–1.0 V and 1.1–1.2 V, attributed to the oxidation of dG and dA, respectively, were registered for the most of oligonucleotides. The oxidation process of ‘dissolved’ (ACTG)n with n = 1–2 via dG was found diffusion-controlled, while (ACTG)3 has demonstrated a mixed mechanism of oxidation reaction. At the same time, ‘adsorbed’ oligonucleotides demonstrated adsorption-limited responses, except for (ACTG)1 showing a mixed reaction mechanism. At a constant nucleotide or oligonucleotide molar concentration, the registered signals for (ACTG)n exponentially decreased with the molecule length. The signals of all studied oligonucleotides after pre-adsorption on electrode surface were up to 10-fold higher than those in the soluble form, except for (ACTG)1. The dG oxidation signal for G-n oligonucleotides, registered at about 0.9 V, was practically insensitive to the position of a single dG (at the end or in the middle of polynucleotide chain), while demonstrating a bell-shaped dependence on the dG amount with a maximum at n = 3–4. The dG oxidation signal for A-n oligonucleotides with a constant portion of dG also increased with the number of dA residues in the bell-shaped fashion with a maximum at n = 5. The folding of oligonucleotides into hairpins with a stem formed via dG/dC pairing resulted in a shift of the peak potential for dG oxidation to higher values. Finally, ultralong ssDNA products generated by rolling circle amplification (RCA) were found to produce strong non-Faradaic current masking the dG and dA oxidation signals. The electrochemical detection of RCA products via dG and dA oxidation was found possible if ultralong ssDNA is fragmented by carrying out RCA in the presence of an appropriate restrictase.