Nucleic Acid Conformation Research Articles

Engineering DNA quadruplexes in DNA nanostructures for biosensor construction.

DNA quadruplexes are nucleic acid conformations comprised of four strands. They are prevalent in human genomes and increasing efforts are being directed toward their engineering. Taking advantage of the programmability of Watson-Crick base-pairing and conjugation methodology of DNA with other molecules, DNA nanostructures of increasing complexity and diversified geometries have been artificially constructed since 1980s. In this review, we investigate the interweaving of natural DNA quadruplexes and artificial DNA nanostructures in the development of the ever-prosperous field of biosensing, highlighting their specific roles in the construction of biosensor, including recognition probe, signal probe, signal amplifier and support platform. Their implementation in various sensing scenes was surveyed. And finally, general conclusion and future perspective are discussed for further developments.

The Simple Biology of Flipons and Condensates Enhances the Evolution of Complexity.

The classical genetic code maps nucleotide triplets to amino acids. The associated sequence composition is complex, representing many elaborations during evolution of form and function. Other genomic elements code for the expression and processing of RNA transcripts. However, over 50% of the human genome consists of widely dispersed repetitive sequences. Among these are simple sequence repeats (SSRs), representing a class of flipons, that under physiological conditions, form alternative nucleic acid conformations such as Z-DNA, G4 quartets, I-motifs, and triplexes. Proteins that bind in a structure-specific manner enable the seeding of condensates with the potential to regulate a wide range of biological processes. SSRs also encode the low complexity peptide repeats to patch condensates together, increasing the number of combinations possible. In situations where SSRs are transcribed, SSR-specific, single-stranded binding proteins may further impact condensate formation. Jointly, flipons and patches speed evolution by enhancing the functionality of condensates. Here, the focus is on the selection of SSR flipons and peptide patches that solve for survival under a wide range of environmental contexts, generating complexity with simple parts.

Submillisecond Conformational Transitions of Short Single-Stranded DNA Lattices by Photon Correlation Single-Molecule Förster Resonance Energy Transfer.

Thermally driven conformational fluctuations (or "breathing") of DNA play important roles in the function and regulation of the "macromolecular machinery of genome expression." Fluctuations in double-stranded (ds) DNA are involved in the transient exposure of pathways to protein binding sites within the DNA framework, leading to the binding of regulatory proteins to single-stranded (ss) DNA templates. These interactions often require that the ssDNA sequences, as well as the proteins involved, assume transient conformations critical for successful binding. Here, we use microsecond-resolved single-molecule Förster resonance energy transfer (smFRET) experiments to investigate the backbone fluctuations of short [oligo(dT)n] templates within DNA constructs that also serve as models for ss-dsDNA junctions. Such junctions, together with the attached ssDNA sequences, are involved in interactions with the ssDNA binding (ssb) proteins that control and integrate the functions of DNA replication complexes. We analyze these data using a chemical network model based on multiorder time-correlation functions and probability distribution functions that characterize the kinetic and thermodynamic behavior of the system. We find that the oligo(dT)n tails of ss-dsDNA constructs interconvert, on submillisecond time scales, between three macrostates with distinctly different end-to-end distances. These are (i) a "compact" macrostate that represents the dominant species at equilibrium; (ii) a "partially extended" macrostate that exists as minority species; and (iii) a "highly extended" macrostate that is present in trace amounts. We propose a model for ssDNA secondary structure that advances our understanding of how spontaneously formed nucleic acid conformations may facilitate the activities of ssDNA-associating proteins.

Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind to nucleic acids.

Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαβDAI), which harbours two Zα domains, and for a ZαβDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαβDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαβDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.

Circular dichroism spectroscopy of DNA duplexes at near-biological concentrations

Circular dichroism (CD) of nucleic acids has been typically carried out at sample concentrations below 10 μM, which is far lower than nucleic acid concentrations in biological systems. Attempts to study nucleic acid conformations by CD at higher concentrations using 10 and 1 mm pathlength cuvettes led to instrument artifacts. By shortening the light pathlength to around 0.1 mm, we herein report the first CD profiles of nucleic acids at sub-mM concentrations, which are relevant to nucleic acid concentrations in cellular cytoplasm and nucleus. These CD experimental conditions will allow future conformational studies of nucleic acids under biologically relevant conditions.

Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

CGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

The biofilm matrix scaffold of Pseudomonas aeruginosa contains G-quadruplex extracellular DNA structures

Extracellular DNA, or eDNA, is recognised as a critical biofilm component; however, it is not understood how it forms networked matrix structures. Here, we isolate eDNA from static-culture Pseudomonas aeruginosa biofilms using ionic liquids to preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions. We describe a loss of eDNA network structure as resulting from a change in nucleic acid conformation, and propose that its ability to form viscoelastic structures is key to its role in building biofilm matrices. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, reveals non-canonical Hoogsteen base pairs, triads or tetrads involving thymine or uracil, and guanine, suggesting that the eDNA forms G-quadruplex structures. These are less abundant in chromosomal DNA and disappear when eDNA undergoes conformation transition. We verify the occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa, as displayed by the matrix to G-quadruplex-specific antibody binding, and validate the loss of G-quadruplex structures in vivo to occur coincident with the disappearance of eDNA fibres. Given their stability, understanding how extracellular G-quadruplex structures form will elucidate how P. aeruginosa eDNA builds viscoelastic networks, which are a foundational biofilm property.

Breaking the Boundary between Solution X-Ray Scattering and Molecular Dynamics Simulations of Structured Nucleic Acids

Nucleic acids perform crucial biological functions in organisms and require specific fold and conformation in the solution. The 3D conformations of nucleic acids are mediated by the solvent condition, sequences, and bound molecules. We investigate fine structural variations of nucleic acids using the full spectrum of solution X-ray scattering, both small-and wide-angle X-ray scattering (SWAXS), and molecular dynamics (MD) simulations. However, integration of SWAXS and MD simulations remains challenging because of 1) issues with conformation sampling, 2) atomically detailed insight without introduced artifacts, and 3) forward modelling of wide-angle profiles from MD simulations. Here, we devise a novel strategy of MD simulations on nucleic acids with enhanced sampling and experimental validations while leaving the underlying physical models intact. We applied this method and revealed the driving forces of structural subtleties in double-stranded and triple-stranded nucleic acid systems.

SRCD and FTIR Spectroscopies to Monitor Protein-Induced Nucleic Acid Remodeling.

Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies are fast techniques providing important information about the conformation of nucleic acids and proteins. These vibrational and electronic absorption spectroscopies are extremely sensitive to any change in molecular structure. While numerous reviews describe how to analyze DNA structure alone or in the presence of proteins using FTIR and CD, analyses of RNA are scarce. Nevertheless, RNA remodeling proteins are important factors involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) and FTIR to analyze how proteins may change RNA structure. These include the analysis of RNA melting, or stabilization, of change in helical parameters and base stacking. The effects on the structure of RNA remodeling proteins are also presented.

Polyamines Mediate Folding of Primordial Hyperacidic Helical Proteins.

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil–helix transitions could be the basis of polyamine regulation in contemporary proteins.

Nucleic Acid Conformation Influences Postsynthetic Suzuki-Miyaura Labeling of Oligonucleotides.

Chemoselective transformations that work under physiological conditions have emerged as powerful tools to label nucleic acids in cell-free and cellular environments. However, detailed studies investigating the influence of nucleic acid conformation on the performance of such chemoselective nucleic labeling methods are less explored. Given that nucleic acids adopt complex structures, it is highly important to study the scope of the chemical modification method in the context of nucleic acid conformations. Here we report a systematic study on the effect of local conformation on the postsynthetic Suzuki-Miyaura functionalization of human telomeric (H-Telo) DNA repeat oligonucleotide (ON) sequences, which form multiple G-quadruplex (GQ) structures. 5-Iodo-2'-deoxyuridine (IdU)-modified H-Telo ONs were synthesized by the solid-phase method, and when subjected to Suzuki-Miyaura cross-coupling reaction, its efficiency was found to depend on the type of conformation and the position of IdU label in different loops of the GQ structure. IdU-labeled GQs gave better yields as compared to single-stranded random coil structures. However, the IdU-labeled duplex under different ionic conditions did not undergo the coupling reaction. Further, using this method, we directly installed an environment-sensitive fluorescent probe, which photophysically reported the formation as well as distinguished different GQ topologies of telomeric repeat. Collectively, this systematic study underscores the influence of nucleic acid conformation, which has to be taken into account when establishing postsynthetic chemoselective functionalization strategies.

CD-NuSS: A Web Server for the Automated Secondary Structural Characterization of the Nucleic Acids from Circular Dichroism Spectra Using Extreme Gradient Boosting Decision-Tree, Neural Network and Kohonen Algorithms

Nucleic acids exhibit a repertoire of conformational preference depending on the sequence and environment. Circular dichroism (CD) is an essential and valuable tool for monitoring such secondary structural conformations of nucleic acids. Nonetheless, the CD spectral diversity associated with these structures poses a challenge in obtaining the quantitative information about the secondary structural content of a given CD spectrum. To this end, the competence of the extreme gradient boosting decision-tree (XGBoost), Kohonen and neural network (nnet) algorithms have been exploited here to predict the diverse secondary structures of nucleic acids. A curated library of 450 CD spectra corresponding to 16 different secondary structures of nucleic acids has been created and used as a training dataset. The hyper-parameters corresponding to the aforementioned algorithms have been optimized using holdout and k-fold (here, k = 5) cross-validation methods. For a test dataset of 150 CD spectra, both the nnet and XGBoost algorithms have exhibited nearly similar prediction accuracy in the range of 85% and 87% (the latter exhibited a slightly higher prediction accuracy). Thus, the nnet and XGBoost algorithms tested here can be employed for predicting the hybrid nucleic acid topologies in future. For the sake of accessibility, the entire process has been automated and implemented as a webserver, called CD-NuSS (CD to nucleic acids secondary structure) and is freely accessible at https://project.iith.ac.in/cdnuss/.

Structural alphabets for conformational analysis of nucleic acids available at dnatco.datmos.org.

A detailed description of the dnatco.datmos.org web server implementing the universal structural alphabet of nucleic acids is presented. It is capable of processing any mmCIF- or PDB-formatted files containing DNA or RNA molecules; these can either be uploaded by the user or supplied as the wwPDB or PDB-REDO structural database access code. The web server performs an assignment of the nucleic acid conformations and presents the results for the intuitive annotation, validation, modeling and refinement of nucleic acids.

Deli Bal ve Grayanotoksin’in Karaciğer Dokusu Üzerindeki Etkilerinin Zamana Bağlı Araştırılması

Amaç: İçindeki grayanotoksin bileşiğinden dolayı insanlarda zehirlenmeye sebep olan deli bal (DB), özellikle Türkiye’nin Karadeniz bölgesinde bazı rahatsızlıkların tedavisinde yaygın olarak kullanılmaktadır. DB zehirlenmesindeki semptomların 1-2 gün içinde normale döndüğü rapor edilmiş olmasına rağmen, iyileşme süresi hakkında ayrıntılı bir çalışma mevcut değildir. Zehirlenme etkilerinin ne kadar süreli olduğunun bilinmesi, hastalara doğru tedavi metodunun uygulanması ve zamanında taburcu edilebilmeleri açısından önemlidir. Bu çalışmanın amacı, etken doz DB ve içindeki aktif maddenin saf hali olan GTX’in karaciğer dokusu üzerindeki toksik etkilerinin zamana bağlı olarak incelenmesidir.Yöntem: Mus musculus türü erkek farelere 75 mg/kg (etken doz) DB ve 0,01 mg/kg GTX-III uygulanmış, bu farelerin 24 ve 48 saat sonra karaciğerleri çıkartılmış ve Azaltılmış Toplam Yansıma-Fourier Dönüşüm Kızılötesi (ATR-FTIR) spektroskopisi ile moleküler düzeyde incelenmiştir.Bulgular: 24 saatlik DB ve GTX uygulaması, karaciğerde lipit peroksidasyonuna, glikojen miktarında azalmaya, doymuş lipit miktarında artışa, membran düzeninde azalmaya, membran akışkanlığında artışa, proteinlerin ve nükleik asitlerin yapı ve konformasyonlarında değişikliklere sebep olmuştur. 48 saatlik süre sonunda glikojen miktarındaki artış her iki grupta, doymuş lipit miktarındaki artış ise sadece DB grubunda normale dönmüştür. Fakat, lipit peroksidasyonu, protein ve nükleik asitlerin yapı ve konformasyonlarındaki, membran düzen ve akışkanlığındaki değişiklikler için 48 saatlik süre yeterli olmamıştır. Sonuç: Sonuç olarak, 24 saatlik DB ve GTX uygulaması karaciğer dokusu üzerinde önemli toksik etkiler oluşturmaktadır ve bu toksik etkilerin hepsinin normale dönmesi için 48 saatlik süre yeterli değildir. Çalışmamızın sonuçları, DB’nin etki süresini ve DB zehirlenmesi vakalarında hastaların tedavi ve taburcu sürelerini belirlemek için faydalı olacaktır.

Application of Synchrotron Radiation Circular Dichroism for RNA Structural Analysis.

Circular dichroism (CD) spectroscopy is a fast and simple technique providing important information about the conformation of nucleic acids, proteins, sugars, lipids, and their interactions between each other. This electronic absorption spectroscopy method is extremely sensitive to any change in molecular structure containing asymmetric molecules. While numerous reviews describe how to analyze deoxyribonucleic acid (DNA) structures using CD, analyses of ribonucleic acids (RNAs) are scarce. Nevertheless, RNAs are important molecules involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) extending the spectral range down to 170nm, improving structural analysis of RNA, including the analysis of helical parameters and alternative structures found in RNA. The effects of temperature to measure thermodynamic parameters and analyze ribonucleoprotein complexes will also be presented.

Guanine Substitutions Prevent Conformational Switch from Antiparallel to Parallel G-Quadruplex.

Guanine quadruplexes, recently reported to form in vivo, represent a broad spectrum of non-canonical conformations of nucleic acids. The actual conformation might differ between water solutions and crowding or dehydrating solutions that better reflect the conditions in the cell. Here we show, using spectroscopic techniques, that most guanine substitutions prevent the conformational switch from antiparallel or hybrid forms to parallel ones when induced by dehydrating agents. The inhibitory effect does not depend on the position of the substitution, but, interestingly, on the type of substitution and, to some extent, on its destabilising potential. A parallel form might be induced in some cases by ligands such as N-methyl mesoporphyrin IX and even this ligand-induced switch is inhibited by guanine substitution. The ability or inability to have a conformation switch, based on actual conditions, might significantly influence potential conformation-dependent quadruplex interactions.

Electrochemical Studies of Cation Condensation-Induced Collapse of Surface-Bound DNA.

In this paper, we demonstrate the ability to control and electrochemically monitor nucleic acid conformation by inducing collapse of short, surface-bound nucleotides (7-28 nucleotides). More specifically, we monitored changes in a 5'-electrode-bound DNA structure via changes in the faradaic current related to the reduction/oxidation of a 3'-terminal-appended redox molecule. Reversible DNA collapse was induced by cation condensation achieved by either reducing the dielectric permittivity of the interrogation solution or by the addition of multivalent cations such as the polyamine spermidine (3+). Additionally, we find that while the change in electrochemical signal associated with surface bound DNA collapse is dependent on nucleic acid length and surface packing density, the solution conditions (e.g., dielectric permittivity) required for collapse remain constant. As such, we find that collapse of the short DNA strands occurs when the effective charge of the DNA backbone is ∼73-89% neutralized by cations in solution/buffer, according to Manning's theory on cation condensation. This work provides new insight into the structure function relationship of surface-bound nucleic acids and how this is manifested in electrochemical signaling.

Insights into telomeric G-quadruplex DNA recognition by HMGB1 protein.

HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.

QPARSE: searching for long-looped or multimeric G-quadruplexes potentially distinctive and druggable.

G-quadruplexes (G4s) are non-canonical nucleic acid conformations that are widespread in all kingdoms of life and are emerging as important regulators both in RNA and DNA. Recently, two new higher-order architectures have been reported: adjacent interacting G4s and G4s with stable long loops forming stem-loop structures. As there are no specialized tools to identify these conformations, we developed QPARSE. QPARSE can exhaustively search for degenerate potential quadruplex-forming sequences (PQSs) containing bulges and/or mismatches at genomic level, as well as either multimeric or long-looped PQS (MPQS and LLPQS, respectively). While its assessment versus known reference datasets is comparable with the state-of-the-art, what is more interesting is its performance in the identification of MPQS and LLPQS that present algorithms are not designed to search for. We report a comprehensive analysis of MPQS in human gene promoters and the analysis of LLPQS on three experimentally validated case studies from HIV-1, BCL2 and hTERT. QPARSE is freely accessible on the web at http://www.medcomp.medicina.unipd.it/qparse/index or downloadable from github as a python 2.7 program https://github.com/B3rse/qparse. Supplementary data are available at Bioinformatics online.

Disordered RNA chaperones can enhance nucleic acid folding via local charge screening

RNA chaperones are proteins that aid in the folding of nucleic acids, but remarkably, many of these proteins are intrinsically disordered. How can these proteins function without a well-defined three-dimensional structure? Here, we address this question by studying the hepatitis C virus core protein, a chaperone that promotes viral genome dimerization. Using single-molecule fluorescence spectroscopy, we find that this positively charged disordered protein facilitates the formation of compact nucleic acid conformations by acting as a flexible macromolecular counterion that locally screens repulsive electrostatic interactions with an efficiency equivalent to molar salt concentrations. The resulting compaction can bias unfolded nucleic acids towards folding, resulting in faster folding kinetics. This potentially widespread mechanism is supported by molecular simulations that rationalize the experimental findings by describing the chaperone as an unstructured polyelectrolyte.