Search

Hydroxyurea (HU; hydroxycarbamide) is a chemotherapy medication used to treat various types of cancer and other diseases such as sickle cell anemia. HU inhibits DNA synthesis by targeting ribonucleotide reductase (RNR). Recent studies have suggested that HU also causes oxidative stress in living systems. In the present study, we investigated if HU could directly affect the activity and/or conformation of DNA. We measured in vitro gene expression in the presence of HU by adapting a cell-free luciferase assay. HU exhibited a bimodal effect on gene expression, where promotion or inhibition were observed at lower or higher concentrations (mM range), respectively. Using atomic force microscopy (AFM), the higher-order structure of DNA was revealed to be partially-thick with kinked-branching structures after HU was added. An elongated coil conformation was observed by AFM in the absence of HU. Single DNA molecules in bulk aqueous solution under fluctuating Brownian motion were imaged by fluorescence microscopy (FM). Both spring and damping constants, mechanical properties of DNA, increased when HU was added. These experimental investigations indicate that HU directly interacts with DNA and provide new insights into how HU acts as a chemotherapeutic agent and targets other diseases., (© 2024. The Author(s).)

The use of insecticides presents a risk to the environment because they can accumulate in the water, soil, air, and organisms, endangering human and animal health. It is therefore essential to investigate the effects of different groups of insecticides on individual biomacromolecules such as DNA. We studied fipronil, which belongs to the group of phenylpyrazole insecticides. The interaction of fipronil with calf thymus DNA was investigated using spectroscopic methods (absorption and fluorescence spectroscopy) complemented with infrared spectroscopy and viscosity measurement. Fluorescence emission spectroscopy showed the formation of a fipronil/DNA complex with a combined static and dynamic type of quenching. The binding constant was 4.15 × 10 3  L/mol. Viscosity changes were recorded to confirm/disconfirm the intercalation mode of interaction. A slight change in DNA viscosity in the presence of fipronil was observed. The phenylpyrazole insecticide does not cause significant conformational changes in DNA structure or increase of its chain length. We hypothesize that fipronil is incorporated into the minor groove of the DNA macromolecule via hydrogen interactions as indicated by FT-IR and CD measurements., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca 2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca 2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca 2+ . Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca 2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing.

Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA-PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na + , while Mg 2+ also played an essential role in the loop formation., (© 2021. The Author(s).)

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg 2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg 2+ -dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

RNAs have become a well-known target for chemotherapeutic agents in the recent years. The tails of most eukaryotic m-RNA are characterized by the presence of a long polyadenylate sequence which plays an important role in its growth and maturation. This lays emphasis on development of molecular probes that target the polyadenylate sequence. Cryptolepine (hereafter, CRP) is an indoloquinoline alkaloid well known for its anti-malarial activities. A series of spectroscopic experiments namely absorption studies, fluorimetric studies and circular dichroism studies show that cryptolepine binds with single-stranded polyriboadenylic acid (hereafter, ss-poly (rA)) with a binding constant of ∼5 × 10 3  M -1  at 25 °C. Moreover thermal denaturation experiments show that the bound form of polyriboadenylic acid shows a characteristic transition profile. Such a profile is indicative of the ability of cryptolepine to induce self-assembly in the polyriboadenylic acid sequence on binding to it. Such ability of CRP to modulate the structural conformation of poly (rA), which in turn may cause functional aspects of the RNA to change, may give us a chance to develop effective alkaloid based chemotherapeutic agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis . We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg 2+ and Sr 2+ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg 2+ . Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The use of a small molecule compound to reduce toxic repeat RNA transcripts or their translated aberrant proteins to target repeat-expanded RNA/DNA with a G4C2 motif is a promising strategy to treat C9orf72-linked disorders. In this study, the crystal structures of DNA and RNA-DNA hybrid duplexes with the -GGGCCG- region as a G4C2 repeat motif were solved. Unusual groove widening and sharper bending of the G4C2 DNA duplex A-DNA conformation with B-form characteristics inside was observed. The G4C2 RNA-DNA hybrid duplex adopts a more typical rigid A form structure. Detailed structural analysis revealed that the G4C2 repeat motif of the DNA duplex exhibits a hydration shell and greater flexibility and serves as a 'hot-spot' for binding of the anthracene-based nickel complex, NiII(Chro)2 (Chro = Chromomycin A3). In addition to the original GGCC recognition site, NiII(Chro)2 has extended specificity and binds the flanked G:C base pairs of the GGCC core, resulting in minor groove contraction and straightening of the DNA backbone. We have also shown that Chro-metal complexes inhibit neuronal toxicity and suppresses locomotor deficits in a Drosophila model of C9orf72-associated ALS. The approach represents a new direction for drug discovery against ALS and FTD diseases by targeting G4C2 repeat motif DNA., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson-Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson-Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The MYCN gene encodes the transcription factor N-Myc, a driver of neuroblastoma (NB). Targeting G-quadruplexes (G4s) with small molecules is attractive strategy to control the expression of undruggable proteins such as N-Myc. However, selective binders to G4s are challenging to identify due to the structural similarity of many G4s. Here, we report the discovery of a small molecule ligand (4) that targets the noncanonical, hairpin containing G4 structure found in the MYCN gene using small molecule microarrays (SMMs). Unlike many G4 binders, the compound was found to bind to a pocket at the base of the hairpin region of the MYCN G4. This compound stabilizes the G4 and has affinity of 3.5 ± 1.6 μM. Moreover, an improved analog, MY-8, suppressed levels of both MYCN and MYCNOS (a lncRNA embedded within the MYCN gene) in NBEB neuroblastoma cells. This work indicates that the approach of targeting complex, hybrid G4 structures that exist throughout the human genome may be an applicable strategy to achieve selectivity for targeting disease-relevant genes including protein coding (MYCN) as well as non-coding (MYCNOS) gene products., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

The involvement of G-quadruplex (G4) structures in nucleic acids in various molecular processes in cells such as replication, gene-pausing, the expression of crucial cancer-related genes and DNA damage repair is well known. The compounds targeting G4 usually bind directly to the G4 structure, but some ligands can also facilitate the G4 folding of unfolded G-rich sequences and stabilize them even without the presence of monovalent ions such as sodium or potassium. Interestingly, some G4-ligand complexes can show a clear induced CD signal, a feature which is indirect proof of the ligand interaction. Based on the dichroic spectral profile it is not only possible to confirm the presence of a G4 structure but also to determine its topology. In this study we examine the potential of the commercially available Rhodamine 6G (RhG) as a G4 ligand. RhG tends to convert antiparallel G4 structures to parallel forms in a manner similar to that of Thiazole Orange. Our results confirm the very high selectivity of this ligand to the G4 structure. Moreover, the parallel topology of G4 can be verified unambiguously based on the specific induced CD profile of the G4-RhG complex. This feature has been verified on more than 50 different DNA sequences forming various non-canonical structural motifs.

Pseudoisocytosine (J), a neutral analogue of protonated cytosine, is currently the gold standard modified nucleobase in peptide nucleic acids (PNAs) for the formation of J·G-C triplets that are stable at physiological pH. This study shows that triple-helical recognition of RNA and DNA is significantly improved by using 2-aminopyridine (M) instead of J. The positively charged M forms 3-fold stronger M+·G-C triplets than J with uncompromised sequence selectivity. Replacement of six Js with Ms in a PNA 9-mer increased its binding affinity by ∼2 orders of magnitude. M-modified PNAs prefer binding double-stranded RNA over DNA and disfavor off-target binding to single-stranded nucleic acids. Taken together, the results show that M is a promising modified nucleobase that significantly improves triplex-forming PNAs and may provide breakthrough developments for therapeutic and biotechnology applications.

Recently, the 1 H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1 H and 19 F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1 H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1 H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19 F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe's discussed limitations, the 19 F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex-ligand interactions in the complex environment of living cells.

Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNA Lys 3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNA Lys 3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

We demonstrated that a synthetic ligand NA, which selectively binds to a 5'-CAG-3'/5'-CAG-3' triad, induced repeat contractions during DNA polymerase-mediated primer extension through the CAG repeat template. A thorough capillary electrophoresis and sequencing analysis revealed that the d(CAG) 20 template gave shortened nascent strands mainly containing 3-6 CTG units in the presence of NA.

Herein, we present a gold nanoparticle (GNP)-based DNA nanosensor to detect the formation of an i-motif from the random coil structure by small molecules at physiological pH. The nanosensor shows a distance dependent fluorescence turn-off response in the presence of a ligand, indicating conformational changes from the C-rich single stranded DNA into an i-motif.

As a naturally occurring class of gene regulators, microRNAs (miRNAs) have attracted much attention as promising targets for therapeutic development. However, RNAs including miRNAs have long been considered undruggable, and most efforts have been devoted to using synthetic oligonucleotides to regulate miRNAs. Encouragingly, recent findings have revealed that miRNAs can also be drugged with small molecules that directly target miRNAs. In this review paper, we give a summary of recently emerged small-molecule inhibitors (SMIs) and small-molecule degraders (SMDs) for miRNAs. SMIs are small molecules that directly bind to miRNAs to inhibit their biogenesis, and SMDs are bifunctional small molecules that upon binding to miRNAs induce miRNA degradation. Strategies for discovering SMIs and developing SMDs were summarized. Applications of SMIs and SMDs in miRNA inhibition and cancer therapy were also introduced. Overall, SMIs and SMDs introduced here have high potency and specificity in miRNA inhibition. We envision that these small molecules will pave the way for developing novel therapeutics toward miRNAs that were previously considered undruggable.

Interactions between aminoglycoside antibiotics and the twister ribozyme were investigated in this study. An initial screen of 17 RNA-binding antibiotics showed that a number of aminoglycosides inhibit the ribozyme, while a subset of aminoglycosides enhances twister cleavage. Initial kinetic analysis of the twister ribozyme showed a sevenfold inhibition of ribozyme cleavage by paromomycin and a fivefold enhancement of cleavage by sisomicin. Direct binding between the twister ribozyme RNA and paromomycin or sisomicin was measured by microscale thermophoresis. Selective 2'-hydroxyl acylation analysed by primer extension shows that both paromomycin and sisomicin induce distinctive tertiary structure changes to the twister ribozyme. Published crystal structures and mechanistic analysis of the twister ribozyme have deduced a nucleobase-mediated general acid-base catalytic mechanism, in which a conserved guanine plays a key role. Here, we show that paromomycin binding induces a structural transition to the twister ribozyme such that a highly conserved guanine in the active site becomes displaced, leading to inhibition of cleavage. In contrast, sisomicin binding appears to change interactions between P3 and L2, inducing allosteric changes to the active site that enhance twister RNA cleavage. Therefore, we show that small-molecule binding can modulate twister ribozyme activity. These results suggest that aminoglycosides may be used as molecular tools to study this widely distributed ribozyme., (© 2020 Federation of European Biochemical Societies.)

Trichothecenes are the most prevalent mycotoxins contaminating cereal grains. Some of them are also considered as the virulence factors of Fusarium head blight disease. However, the mechanism behind the structure-activity relationship for trichothecenes remains unexplained. Filling this information gap is a crucial step for developing strategies to manage this large family of mycotoxins in food and feed. Here, we perform an in-depth re-examination of the existing structures of Saccharomyces cerevisiae ribosome complexed with three different trichothecenes. Multiple binding interactions between trichothecenes and 25S rRNA, including hydrogen bonds, nonpolar pi stacking interactions and metal ion coordination interactions, are identified as important binding determinants. These interactions are mainly contributed by the key structural elements to the toxicity of trichothecenes, including the oxygen in the 12,13-epoxide ring and a double bond between C 9 and C 10 . In addition, the C 3 -OH group also participates in binding. The comparison of three trichothecenes binding to the ribosome, along with their binding pocket architecture, suggests that the substitutions at different positions impact trichothecenes binding in two different patterns. Moreover, the binding of trichothecenes induced conformation changes of several nucleotide bases in 25S rRNA. This then provides a structural framework for understanding the structure-activity relationships apparent in trichothecenes. This study will facilitate the development of strategies aimed at detoxifying mycotoxins in food and feed and at improving the resistance of cereal crops to Fusarium fungal diseases.

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C 8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C 8 , demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.

Background: Several different small molecules have been used to target the DNA helix in order to treat the diseases caused by its mutation. Guanidinium(Gdm + ) and urea based drugs have been used for the diseases related to central nervous system, also as the anti-inflammatory and chemotherapeutic agent. However, the role of Gdm + and urea in the stabilization/destabilization of DNA is not well understood., Methods: Spectroscopic techniques along with molecular dynamics (MD) simulation have been performed on different sequences of DNA in the presence of guanidinium chloride (GdmCl) and urea to decode the binding of denaturants with DNA and the role of hydrogen bond with the different regions of DNA in its stability/destability., Results and Conclusion: Our study reveals that, Gdm + of GdmCl and urea both intrudes into the groove region of DNA along with the interaction with its phosphate backbone. However, interaction of Gdm + and urea with the nucleobases in the groove region is different. Gdm + forms the intra-strand hydrogen bond with the central region of the both sequences of DNA whereas inter-strand hydrogen bond along with water assisted hydrogen bond takes place in the case of urea. The intra-strand hydrogen bond formation capability of Gdm + with the nucleobases in the minor groove of DNA decreases its groove width which probably causes the stabilization of B-DNA in GdmCl. In contrast, the propensity of the formation of inter-strand hydrogen bond of urea with the nucleobases in the groove region of DNA without affecting the groove width destabilizes B-DNA as compared to GdmCl. This study depicts that the opposite effect of GdmCl and urea on the stability is a general property of B-DNA. However, the extent of stabilization/destabilization of DNA in Gdm + and urea depend on its sequence probably due to the difference in the intra/inter-strand hydrogen bonding with different bases present in both the sequences of DNA., General Significance: The information obtained from this study will be useful for the designing of Gdm + based drug molecule which can target the DNA more specifically and selectively., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Peroxynitrite is known to react with biomolecules leading to their structural and function alteration. Structural alteration in DNA induced by peroxynitrite is not clearly known. The current study was carried out to decipher the changes induced by peroxynitrite using UV-Vis spectra, circular dichrometry, molecular dynamics simulation followed by restriction digestion. Apoptotic markers Bax, Bcl-2 and caspase genes were also studied by FACS in peroxynitrite induced PC12 cells. The results obtained showed that PXN binds to DNA leading to hyperchromicity of DNA in the presence of PXN over a period of time and the same was established by In silico studies where PXN modifies the DNA to accommodate itself into the stacking and brings about the significant structural alterations. Further, FACS studies reveal that Bcl-2 gene expression was down regulated whereas BAXand caspase genes were up regulated compared to control concluding that PXN induces apoptotic cell death in PC12 cells.

Acetamiprid (ACE) is a kind of broad-spectrum pesticide that has potential health risk to human beings. Aptamers (Ap-DNA (1)) have a great potential as analytical tools for pesticide detection. In this work, a label-free electrochemical sensing assay for ACE determination is presented by electrochemical impedance spectroscopy (EIS). And the specific binding model between ACE and Ap-DNA (1) was further investigated for the first time. Circular dichroism (CD) spectroscopy and EIS demonstrated that the single strand AP-DNA (1) first formed a loosely secondary structure in Tris-HClO4 (20 mM, pH = 7.4), and then transformed into a more stable hairpin-like structure when incubated in binding buffer (B-buffer). The formed stem-loop bulge provides the specific capturing sites for ACE, forming ACE/AP-DNA (1) complex, and induced the RCT (charge transfer resistance) increase between the solution-based redox probe [Fe(CN)6]3-/4- and the electrode surface. The change of ΔRCT (charge transfer resistance change, ΔRCT = RCT(after)-RCT(before)) is positively related to the ACE level. As a result, the AP-DNA (1) biosensor showed a high sensitivity with the ACE concentration range spanning from 5 nM to 200 mM and a detection limit of 1 nM. The impedimetric AP-DNA (1) sensor also showed good selectivity to ACE over other selected pesticides and exhbited excellent performance in environmental water and orange juice samples analysis, with spiked recoveries in the range of 85.8% to 93.4% in lake water and 83.7% to 89.4% in orange juice. With good performance characteristics of practicality, sensitivity and selectivity, the AP-DNA (1) sensor holds a promising application for the on-site ACE detection., Competing Interests: JH Zhen and RC Chen are affiliated with Shijiazhuang Customs Technology Center. There are no patents, products in development or marketed products to declare. G Liang and WS Jia are affiliated with Beijing Research Center for Agricultural Standards and Testing, Beijing Academy of Agriculture and Forestry Science. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Pyrrole-imidazole (Py-Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide-DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles., Competing Interests: DB is a sole proprietor of Bio Informaticals (see www.bioinformaticals.com). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

RNA structure is critically important to RNA viruses in every part of the replication cycle. RNA structure is also utilized by DNA viruses in order to regulate gene expression and interact with host factors. Advances in next-generation sequencing have greatly enhanced the utility of chemical probing in order to analyze RNA structure. This review will cover some recent viral RNA structural studies using chemical probing and next-generation sequencing as well as the advantages of dimethyl sulfate (DMS)-mutational profiling and sequencing (MaPseq). DMS-MaPseq is a robust assay that can easily modify RNA in vitro, in cell and in virion. A detailed protocol for whole-genome DMS-MaPseq from cells transfected with HIV-1 and the structure of TAR as determined by DMS-MaPseq is presented. DMS-MaPseq has the ability to answer a variety of integral questions about viral RNA, including how they change in different environments and when interacting with different host factors., (Published by Elsevier Inc.)

In nature, allostery is the principal approach for regulating cellular processes and pathways. Inspired by nature, structure-switching aptamer-based nanodevices are widely used in artificial biotechnologies. However, the canonical aptamer structures in the nanodevices usually adopt a duplex form, which limits the flexibility and controllability. Here, a new regulating strategy based on a clamp-like triplex aptamer structure (CLTAS) was proposed for switching DNA polymerase activity via conformational changes. It was demonstrated that the polymerase activity could be regulated by either adjusting structure parameters or dynamic reactions including strand displacement or enzymatic digestion. Compared with the duplex aptamer structure, the CLTAS possesses programmability, excellent affinity and high discrimination efficiency. The CLTAS was successfully applied to distinguish single-base mismatches. The strategy expands the application scope of triplex structures and shows potential in biosensing and programmable nanomachines., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here we report that incorporation of an abasic site to DNA minidumbbells formed by natural sequences can lead to significant enhancements in their thermodynamic stability. Based on these stable minidumbbells, the first metal ion-controlled molecular switch which can regulate instant and reversible DNA duplex formation and dissociation has been constructed.

In this work, using [Ru(bpy) 2 (pip)] 2+ (bpy = 2,2'-bipyridine, pip = 2-phenyl-1H-imidazo[4,5-f]-[1,10]-phenanthroline) as chromophores and neutral amino acid glycine as spacers, two novel Arg- and Lys-rich Ru(II) polypyridyl metallopeptides as an intermolecular triplex RNA stabilizers, namely [Ru(bpy) 2 (pic-Lys 2 -Gly-Lys 2 -Gly-Lys 2 )] 8+ (Ru1; pic = 2-(4-carboxy-phenyl)imidazo-[4,5-f] [1,10] phenanthroline, Gly = glycine, Lys = lysine) and [Ru(bpy) 2 (pic-Arg 2 -Gly-Arg 2 -Gly-Arg 2 )] 8+ (Ru2; Arg = arginine), have been synthesized and characterized. The binding properties of Ru1 and Ru2 with poly(U)·poly(A)∗poly(U) triplex have been studied by UV-Vis spectroscopy, fluorescence spectroscopy, viscosity measurements as well as circular dichroism and thermal denaturation. The obtained results suggest that attaching cationic peptides to a Ru(II) polypyridyl complex can obviously enhance the triplex stabilization. Considering the structure natures of Ru1 and Ru2, conceivably besides electrostatic interaction, the forces stabilizing the triplex should also involve hydrophobic interaction and hydrogen binding. Compared with the Lys-rich metallopeptide (Ru1), however, the third-strand stabilizating effect of the Arg-rich one (Ru2) is slightly more marked, which may be due to differences in the interactions of arginine and lysine residues with the third strand of the triplex. The results obtained here may be useful for understanding the interaction of triplex RNA poly(U)·poly(A)∗poly(U) with small molecule, particularly ruthenium(II) complexes., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2020 Elsevier Inc. All rights reserved.)

DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aberrant RNA structure and function operate in neurological disease progression and severity. As RNA contributes to disease pathology in a complex fashion, that is, via various mechanisms, it has become an attractive therapeutic target for small molecules and oligonucleotides. In this review, we discuss the identification of RNA structures that cause or contribute to neurological diseases as well as recent progress toward the development of small molecules that target them, including small molecule modulators of pre-mRNA splicing and RNA repeat expansions that cause microsatellite disorders such as Huntington's disease and amyotrophic lateral sclerosis. The use of oligonucleotide-based modalities is also discussed. There are key differences between small molecule and oligonucleotide targeting of RNA. The former targets RNA structure, while the latter prefers unstructured regions. Thus, some targets will be preferentially targeted by oligonucleotides and others by small molecules., (© 2019 New York Academy of Sciences.)

Herein we present the synthesis of a l-diaminobutanoic acid (DABA)-based nucleopeptide (3), with an oligocationic backbone, realized by solid phase peptide synthesis using thymine-bearing DABA moieties alternating in the sequence with free ones. CD studies evidenced the ability of this oligothymine nucleopeptide, well soluble in aqueous solution, to alter the secondary structure particularly of complementary RNA (poly rA vs poly rU) and inosine-rich RNAs, like poly rI and poly rIC, and showed its preference in binding double vs single-stranded DNAs. Furthermore, ESI mass spectrometry revealed that 3 bound also G-quadruplex (G4) DNAs, with either parallel or antiparallel topologies (adopted in our experimental conditions by c-myc and tel 22 , respectively). However, it caused detectable changes only in the CD of c-myc (whose parallel G4 structure was also thermally stabilized by ~3 °C), while leaving unaltered the antiparallel structure of tel 22 . Interestingly, CD and UV analyses suggested that 3 induced a hybrid mixed parallel/antiparallel G4 DNA structure in a random-coil tel 22 DNA obtained under salt-free buffer conditions. Titration of the random-coil telomeric DNA with 3 gave quantitative information on the stoichiometry of the obtained complex. Overall, the findings of this work suggest that DABA-based nucleopeptides are synthetic nucleic acid analogues potentially useful in antigene and antisense strategies. Nevertheless, the hexathymine DABA-nucleopeptide shows an interesting behaviour as molecular tool per se thanks to its efficacy in provoking G4 induction in random coil G-rich DNA, as well as for the possibility to bind and stabilize c-myc oncogene in a G4 structure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops-c-shapes or s-shapes-that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Double-stranded oligonucleotides containing cisplatin adducts, with and without a mismatched region, were exposed to hydrated electrons generated by gamma-rays. Gel electrophoresis analysis demonstrates the formation of cisplatin-interstrand crosslinks from the cisplatin-intrastrand species. The rate constant per base for the reaction between hydrated electrons and the double-stranded oligonucleotides with and without cisplatin containing a mismatched region was determined by pulse radiolysis to be 7 × 109 and 2 × 109 M-1 s-1, respectively. These results provide a better understanding of the radiosensitizing effect of cisplatin adducts in hypoxic tumors and of the formation of interstrand crosslinks, which are difficult for cells to repair., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

G-quadruplex, assembled from a square array of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a versatile template for ordered functional materials. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking modes and the spontaneous orientation of G-quadruplex fibrils. Here, the effects of different metal ions and their concentrations on stacking modes of G-quartets are elucidated. Monovalent cations (typically K + ) facilitate the formation of G-quadruplex hydrogels with both heteropolar and homopolar stacking modes, showing weak mechanical strength. In contrast, divalent metal ions (Ca 2+ , Sr 2+ , and Ba 2+ ) at given concentrations can control G-quartet stacking modes and increase the mechanical rigidity of the resulting hydrogels through ionic bridge effects between divalent ions and borate. We show that for Ca 2+ and Ba 2+ at suitable concentrations, the assembly of G-quadruplexes results in the establishment of a mesoscopic chirality of the fibrils with a regular left-handed twist. Finally, we report the discovery of nematic tactoids self-assembled from G-quadruplex fibrils characterized by homeotropic fibril alignment with respect to the interface. We use the Frank-Oseen elastic energy and the Rapini-Papoular anisotropic surface energy to rationalize two different configurations of the tactoids. These results deepen our understanding of G-quadruplex structures and G-quadruplex fibrils, paving the way for their use in self-assembly and biomaterials., Competing Interests: The authors declare no competing interest.

We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2'-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL-G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.

Two novel electrochemiluminescence (ECL) deoxyribosensors are designed for assay of early lung cancer biomarker (NAP2) using the DNA three-way junction (DNA-TWJ) inserted NAP2 binding aptamer between two double-helical stems and labeled with ruthenium (II) complex (Ru) (NBAT-Ru) taken as molecular recognition element. The signal-off ECL deoxyribosensor was fabricated by covalently coupling the 5'-NH 2 -(CH 2 ) 6 -NBAT-Ru to glassy carbon electrode surface modified with 4-aminobenzoic acid (4-ABA). After combining NAP2 and NBAT-Ru, the changed conformation of NBAT-Ru altered the distance between Ru complex and electrode, resulting in a low ECL signal. The signal-on deoxyribosensor was fabricated by self-assembling the 5'-SH-(CH 2 ) 6 -NBAT-Ru onto the Au electrode. The introduction of NAP2 triggered the conformational change in the aptamer domain, which induces the interhelical stacking of the two double-helical stems of NBAT-Ru. This stacking constitutes "electrical contact," which promotes transmission of electron-holes through the stems of NBAT-Ru, and produces high ECL intensity. Both deoxyribosensors show high sensitivity and selectivity. The biosensors have been successfully applied to clinical plasma detection. The approaches we describe represent unique principles based on DNA-TWJ inserted target special binding domain as molecular recognition element and different immobilization types for the fabrication of biosensors, which are greatly promising for the detection of protein, metal ions, bacteria, and cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The use of nanoparticles (NP) as dose enhancers in radiotherapy (RT) is a growing research field. Recently, the use of NP has been extended to charged particle therapy in order to improve the performance in radioresistant tumors. However, the biological mechanisms underlying the synergistic effects involved in NP-RT approaches are not clearly understood. Here, we used the capabilities of synchrotron-based Fourier Transform Infrared Microspectroscopy (SR-FTIRM) as a bio-analytical tool to elucidate the NP-induced cellular damage at the molecular level and at a single-cell scale. F98 glioma cells doped with AuNP and GdNP were irradiated using several types of medical ion beams (proton, helium, carbon and oxygen). Differences in cell composition were analyzed in the nucleic acids, protein and lipid spectral regions using multivariate methods (Principal Component Analysis, PCA). Several NP-induced cellular modifications were detected, such as conformational changes in secondary protein structures, intensity variations in the lipid CHx stretching bands, as well as complex DNA rearrangements following charged particle therapy irradiations. These spectral features seem to be correlated with the already shown enhancement both in the DNA damage response and in the reactive oxygen species (ROS) production by the NP, which causes cell damage in the form of protein, lipid, and/or DNA oxidations. Vibrational features were NP-dependent due to the NP heterogeneous radiosensitization capability. Our results provided new insights into the molecular changes in response to NP-based RT treatments using ion beams, and highlighted the relevance of SR-FTIRM as a useful and precise technique for assessing cell response to innovative radiotherapy approaches.

Background: Denaturants, namely, urea and guanidinium chloride (GdmCl) affect the stability as well as structure of DNA. Critical assessment of the role of hydrogen bonding of these denaturants with the different regions of DNA is essential in terms of its stability and structural aspect. However, the understanding of the mechanistic aspects of structural change of DNA induced by the denaturants is not yet well understood., Methods: In this study, various spectroscopic along with molecular dynamics (MD) simulation techniques were employed to understand the role of hydrogen bonding of these denaturants with DNA bases in their stability and structural change., Results and Conclusion: It has been found that both, GdmCl and urea intrude into groove region of DNA by striping surrounding water. The hydrogen bonding pattern of Gdm + and urea with DNA bases in its groove region is multimodal and distinctly different from each other. The interaction of GdmCl with DNA is stabilized by electrostatic interaction whereas electrostatic and Lennard-Jones interactions both contribute for urea. Gdm + forms direct hydrogen bond with the bases in the minor groove of DNA whereas direct and water assisted hydrogen bond takes place with urea. The hydrogen bond formed between Gdm + with bases in the groove region of DNA is stronger than urea due to strong electrostatic interaction along with less self-aggregation of Gdm + than urea. The distinct hydrogen bonding capability of Gdm + and urea with DNA bases in its groove region affects its width differently. The interaction of Gdm + decreases the width of the minor and major groove which probably increases the strength of hydrogen bond between the Watson-Crick base pairs of DNA leading to its stability. In contrast, the interaction of urea does not affect much to the width of the grooves except the marginal increase in the minor groove width which probably decreases the strength of hydrogen bond between Watson Crick base pairs leading to the destabilization of DNA., General Significance: Our study clearly depicts the role of hydrogen bonding between DNA bases and denaturants in their stability and structural change which can be used further for designing of the guanidinium based drug molecules., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Riboswitches alter gene expression in response to ligand binding, coupling sensing and regulatory functions to help bacteria respond to their environment. The structural determinants of ligand binding in the prequeuosine (7-aminomethyl-7-deazaguanine, preQ 1 ) bacterial riboswitches have been studied, but the functional consequences of structural perturbations are less known. A new article combining biophysical and cell-based readouts of 15 mutants of the preQ 1 -II riboswitch from Lactobacillus rhamnosus demonstrates that ligand binding does not ensure successful gene regulation, providing new insights into these shapeshifting sequences., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kierzek and Kierzek.)

Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Near infra-red (NIR) light emitting nanomaterials had shown great promise in clinical imaging in view of negligible absorption by skin or tissue of mammalian. Thus, it demands for synthesizing stable NIR emitting nanomaterials in water environment. The present work presents synthesis of biologically acceptable luminescent near-IR emitting silver sulfide nanoparticles through an aqueous route using 2-mercaptoethanol. The prepared as-synthesized Ag 2 S nanoparticles exhibited bright photoluminescence with quantum yield of ca. 4%. X-ray diffraction (XRD) analysis indicated that the products were monoclinic α-Ag 2 S. Fourier transform infrared spectral analysis revealed that the stretching vibration at 2560 cm -1 responsible for S-H bond of thiol group disappeared suggesting the conjugation of 2-mercaptoethanol with Ag 2 S nanoparticles. In view of investigating any possible effect on genetic materials, interactions of the synthesized particles with calf thymus DNA was investigated employing Ethidium bromide (EB) as structural probe. To understand the binding mechanism, the UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopic, as well as DNA melting studies measurements were carried out. The observed results confirm that nanoparticles interact with DNA through groove binding., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The properties of non-canonical DNA structures, like G-quadruplexes and triplexes, change under cell-mimicking molecular crowding conditions relative to dilute aqueous solutions. The analysis of environmental effects on their stability is crucial since they play important roles in gene expression and regulation. In this study, three intramolecular and intermolecular triplex-forming sequences of different C + * G-C triplet content ( * : Hoogsteen base pair; - : Watson-Crick base pair) were designed and their stability measured in the absence and presence of a crowding agent with different K + concentrations. In dilute solution, the stability of the triplexes was reduced by decreasing the concentration of KCl. This reduction became smaller as the number of C + * G-C triplets increased. Under molecular crowding conditions, Watson-Crick base pairs and Hoogsteen base pairs were destabilized and stabilized, respectively. Interestingly, with lower KCl concentrations (≤1 M), the destabilization of the triplexes due to reduction of KCl concentration was significantly smaller than in dilute solutions. In addition, the C + * G-C content had greater influence on triplex stability under molecular crowding conditions. Our work provides quantitative information about the effects of K + concentration on triplex stability under molecular crowding conditions and should further our understanding of the function and regulation of triplexes in bioprocesses.

The mode of action of Pt- and Pd-based anticancer agents (cisplatin and Pd 2 Spm) was studied by characterising their impact on DNA. Changes in conformation and mobility at the molecular level in hydrated DNA were analysed by quasi-elastic and inelastic neutron scattering techniques (QENS and INS), coupled to Fourier transform infrared (FTIR) and microRaman spectroscopies. Although INS, FTIR and Raman revealed drug-triggered changes in the phosphate groups and the double helix base pairing, QENS allowed access to the nanosecond motions of the biomolecule's backbone and confined hydration water within the minor groove. Distinct effects were observed for cisplatin and Pd 2 Spm, the former having a predominant effect on DNA´s spine of hydration, whereas the latter had a higher influence on the backbone dynamics. This is an innovative way of tackling a drug´s mode of action, mediated by the hydration waters within its pharmacological target (DNA)., Competing Interests: The authors declare no conflict of interest.

DNA is an essential target for the treatment of various pathologies, especially cancer. Hence targeting DNA double helix for alteration of its function has been attempted by several ways. Drug-DNA intercalation, one such biophysical process, could not be studied extensively as this requires significant deformation of the receptor DNA. Here we report thorough theoretical investigation of intercalation process in daunomycin-DNA interaction, by performing molecular dynamics simulations of the drug-DNA complexes for various DNA sequences, followed by Free-energy analysis and density functional theory (DFT) based studies to understand the binding preference. The classical energy based analyses indicate that the drug prefers to bind to TC/GA sequence over others. The DFT based energies of supra-molecular complexes are always contaminated with basis set superposition error (BSSE), which can be corrected by counterpoise method. This method is quite effective for systems containing two molecular fragments but is not appropriate for studying interaction between two base pair fragments and the drug intercalated between them. We have adopted an extension of the counterpoise method for BSSE corrected interaction energy calculation. These interaction energies, along with the energy penalty due to un-stacking of the base pairs, also indicate TC/GA sequence is the most preferred sequence for binding.

Oligonucleotide drugs (ODs) have gained increasing attention owing to their promising therapeutic potential. One major obstacle that ODs have been facing is the lack of appropriate in vitro validation systems that can predict in vivo activity and toxicity. We have devised a transfection method called CEM (Ca 2+ -enrichment method), where the simple enrichment of calcium ion with calcium chloride in culture medium potentiates the activity of various types of naked oligonucleotides including gapmers, siRNA, and phosphorodiamidate morpholino antisense oligonucleotides (PMO) in many cultured cell lines with limited cytotoxicity. We here describe a precise procedure of the method. Besides the benefit of the CEM's predictive power to accurately estimate in vivo activity of ODs of your interest in drug discovery and development settings, this cost-efficient, easy-to-access method can be a robust laboratory technique to modulate gene expressions with ODs with a variety of mechanisms of action.