Terminal Base Pairs Research Articles

Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

Theta-mediated end-joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend single-stranded DNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both single-stranded DNA and RNA substrates by unimolecular stem loop synthesis initiated by only two 3ʹ terminal base-pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem loop synthesis competes with bimolecular end-joining, even when a longer terminal microhomology for end-joining is available. Both reactions are partially suppressed by the ssDNA binding protein RPA. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem-loop synthesis. The ability to perform stem-loop synthesis from a minimally paired primer is rare amongst human DNA polymerases but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.

Length-independent and selective sensing of CCG trinucleotide repeat foldings by a monovalency-binding fluorogenic probe

Tandem CNG (N = C, T, A, G) DNA trinucleotide repeats (TNRs) are the main expansion to cause severe neurodegenerative diseases. Small molecule ligands with a capacity to sense the CNG TNRs foldings have received considerable attention because of therapeutic and diagnostic perspectives. This requires development of effectual ligands that have a preference to a particular CNG TNRs foldings. However, the previously developed ligands using hydrogen-bonding interaction as the dominantly driving force usually exhibit a multimolecular binding behavior with TNRs. This multivalency interaction usually compromises the sensing selectivity towards CNG TNRs. Herein, we reported a monovalency probe of pseudohypericin (PHP) to selectively sense only the CCG TNRs foldings by a signal-on fluorescence, while total signal-off responses were otherwise observed for CTG, CAG, and CGG TNRs. In addition, the monovalency association of PHP to CCG TNRs is independent of the repeat length. This selective sensing occurs in the loop region, in which a triad is formed between the terminal Watson-Crick CG base pair and the mismatched cytosine adopting an extrahelical conformation. We believe that the specific interaction of PHP with the triad is in charge of the selective association of CCG TNRs over other counterparts. Furthermore, the sensitivity to CCG TNRs can be strengthened by the repeat-activated photocatalysis activity of the TNRs-based DNAzymes via a singlet oxygen (1O2) pathway. Our work offers an alternative way to sense CNG TNRs with a high selectivity and promising applications in developing TNRs-based sensors.

Stability of End-to-End Base Stacking Interactions in Highly Concentrated DNA Solutions.

Positionally ordered bilayer liquid crystalline nanostructures formed by gapped DNA (GDNA) constructs provide a practical window into DNA-DNA interactions at physiologically relevant DNA concentrations; concentrations several orders of magnitude greater than those in commonly used biophysical assays. The bilayer structure of these states of matter is stabilized by end-to-end base stacking interactions; moreover, such interactions also promote in-plane positional ordering of duplexes that are separated from each other by less than twice the duplex diameter. The end-to-end stacked as well as in-plane ordered duplexes exhibit distinct signatures when studied via small-angle X-ray scattering (SAXS). This enables analysis of the thermal stability of both the end-to-end and side-by-side interactions. We performed synchrotron SAXS experiments over a temperature range of 5-65 °C on GDNA constructs that differ only by the terminal base-pairs at the blunt duplex ends, resulting in identical side-by-side interactions, while end-to-end base stacking interactions are varied. Our key finding is that bilayers formed by constructs with GC termination transition into the monolayer state at temperatures as much as 30 °C higher than for those with AT termination, while mixed (AT/GC) terminations have intermediate stability. By modeling the bilayer melting in terms of a temperature-dependent reduction in the average fraction of end-to-end paired duplexes, we estimate the stacking free energies in DNA solutions of physiologically relevant concentrations. The free-energies thereby determined are generally smaller than those reported in single-molecule studies, which might reflect the elevated DNA concentrations in our studies.

Structural basis of tRNAPro acceptor stem recognition by a bacterial trans-editing domain.

High fidelity tRNA aminoacylation by aminoacyl-tRNA synthetases is essential for cell viability. ProXp-ala is a trans-editing protein that is present in all three domains of life and is responsible for hydrolyzing mischarged Ala-tRNAPro and preventing mistranslation of proline codons. Previous studies have shown that, like bacterial prolyl-tRNA synthetase, Caulobacter crescentus ProXp-ala recognizes the unique C1:G72 terminal base pair of the tRNAPro acceptor stem, helping to ensure deacylation of Ala-tRNAPro but not Ala-tRNAAla. The structural basis for C1:G72 recognition by ProXp-ala is still unknown and was investigated here. NMR spectroscopy, binding, and activity assays revealed two conserved residues, K50 and R80, that likely interact with the first base pair, stabilizing the initial protein-RNA encounter complex. Modeling studies are consistent with direct interaction between R80 and the major groove of G72. A third key contact between A76 of tRNAPro and K45 of ProXp-ala was essential for binding and accommodating the CCA-3' end in the active site. We also demonstrated the essential role that the 2'OH of A76 plays in catalysis. Eukaryotic ProXp-ala proteins recognize the same acceptor stem positions as their bacterial counterparts, albeit with different nucleotide base identities. ProXp-ala is encoded in some human pathogens; thus, these results have the potential to inform new antibiotic drug design.

Refreshing DNA-Based Nanoprobes by Alcohol for Heavy Metal Detection: Toward Sustainable Sensing Nanomaterials

Increasing the applications of disposable nanomaterials yet with potential environmental risk and the resulting nanowastes have become a problem of public concern worldwide. However, efficient recovery of useful materials from nanowastes is currently limited by their tiny size, high physicochemical activity, and unpredictable aggregation. We have developed a conceptually new strategy for recycling nanoprobes by alcoholic solvent “refreshing”. In specific, DNA-functionalized gold nanoparticles and nanorods are employed as colorimetric nanoprobes for Hg(II) and Ag(I) ions, respectively, both of which rely upon target-induced DNA terminal base pairing and the subsequent base pair stacking assembly of the nanoprobes. Upon the introduction of isopropanol to the “waste” aggregates, however, the DNAs are denaturalized to release heavy metal ions, leading to restoration of individual nanoprobes. The fine modulation of DNA hybridization not only affords sensitive detection but also addresses the recycling of the nanoprobes with high efficiency (>80%) while preserving the detection performance irrespective of the nanoprobe shape and surface-grafted DNA sequence. We further demonstrate the use of the recycled nanoprobes in the detection of Hg(II) species from salmon muscle tissue. With the generality of the recovering method and the combined features of the renewable nanoprobes, including high recycling efficiency and retained functionality, sustainable sensing nanomaterials are within reach.

Molecular insights into the stereospecificity of arginine in RNA tetraloop folding.

One of the hypotheses for the homochirality of amino acids in the context of the origin of life is that only a particular stereoisomer provides preferential stability to RNA folding by acting as a chemical chaperon. However, the effect at the molecular level is not well understood. This study provides a molecular understanding of such preferential stability for a small GAAA RNA tetraloop in the presence of chiral arginine through a multidimensional free energy landscape constructed using a combination of umbrella sampling and parallel bias metadynamics (PBMetaD) simulations. We show that the origin of the chirality difference in RNA folding-unfolding dynamics is due to differences in the configurational diversity of RNA in adopting various non-natural conformations that accompany the diverse binding modes of D-arginine and L-arginine. We show that while D-arginine stabilizes the native folded state of RNA, L-arginine destabilizes it. Furthermore, free energy calculations on the binding of D- and L-arginine reveal a specific geometric constraint that helps D-arginine to stack with the terminal base pairs of RNA and pushes L-arginine for groove binding.

19F electron nuclear double resonance (ENDOR) spectroscopy for distance measurements using trityl spin labels in DNA duplexes.

The combination of fluorine labeling and pulsed electron-nuclear double resonance (ENDOR) is emerging as a powerful technique for obtaining structural information about proteins and nucleic acids. In this work, we explored the capability of Mims 19F ENDOR experiments on reporting intermolecular distances in trityl- and 19F-labeled DNA duplexes at three electron paramagnetic resonance (EPR) frequencies (34, 94, and 263 GHz). For spin labeling, we used the hydrophobic Finland trityl radical and hydrophilic OX063 trityl radical. Fluorine labels were introduced into two positions of a DNA oligonucleotide. The results indicated that hyperfine splittings visible in the ENDOR spectra are consistent with the most populated interspin distances between 19F and the trityl radical predicted from molecular dynamic (MD) simulations. Moreover, for some cases, ENDOR spectral simulations based on MD results were able to reproduce the conformational distribution reflected in the experimental ENDOR line broadening. Additionally, MD simulations provided more detailed information about the melting of terminal base pairs of the oligonucleotides and about the configuration of the trityls relative to a DNA end.

Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

Soot and charcoal as reservoirs of extracellular DNA

The vast potential of using sediment adsorbed DNA as a window to past and present biodiversity rely on the ability of solid surfaces to adsorb environmental DNA. However, a comprehensive insight into DNA adsorption at surfaces in general is lacking. Soot and charcoal are carbonaceous materials widespread in the environment where they readily can come in contact with extracellular DNA shed from organisms. Using batch adsorption, we measured DNA adsorption capacity at soot and charcoal as a function of solution composition, time and DNA length. We observed that the adsorption capacity for DNA is highest at low pH, that it increases with solution concentration and cation valency and that the activation energy for DNA adsorption at both soot and charcoal is ~50 kJmol-1, suggesting strong binding. We demonstrate how the interaction between DNA and soot and charcoal partly occurs via terminal base pairs, suggesting that, besides electrostatic forces, hydrophobic interactions play an important role in binding. The large adsorption capacities and strong binding of DNA to soot and charcoal are features important for eDNA research and provide a motivation for use of carbonaceous materials from, e.g., anthropogenic pollution or wildfire as sources of biodiversity information.

Nearest neighbor rules for RNA helix folding thermodynamics: improved end effects.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5′GGUC3′ paired to 3′CUGG5′ separately from other 5′GU3′/3′UG5′ stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

DNA-Dependent Binding of Nargenicin to DnaE1 Inhibits Replication in Mycobacterium tuberculosis.

Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.

Orientation Dependence of DNA Blunt-End Stacking Studied by Free-Energy Simulations

DNA blunt ends can associate mediated by stacking interactions between the terminal base pairs that form blunt ends. The blunt end association plays a role in DNA repair and recombination processes and can also be of importance for the design of DNA-based nano-materials. Its function depends on the sequence and on the geometric arrangement that leads to stable interaction. For a stacked state, the relative orientation (twisting) of the base pairs is important. Molecular dynamics and advanced sampling simulations were used to calculate free energy change associated with twist changes of the stacked blunt-end base pairs. The calculations reproduce blunt stacking arrangements found in crystal structures of DNA oligonucleotides as free energy minima. To elucidate the physical origin of the stabilization of certain angular arrangements, the interactions between backbone atoms in the blunt-end stack were switched off in additional free energy calculations. It allows us to decipher the contributions to stacking stabilization due to the nucleobases and the backbone and to analyze the sequence dependence of the angular stacking preferences. Good qualitative agreement was also found for the comparison with quantum mechanical calculations. The results may help in the design of novel DNA-based materials.

Membrane Activity of a DNA-Based Ion Channel Depends on the Stability of Its Double-Stranded Structure.

DNA nanotechnology has emerged as a promising method for designing spontaneously inserting and fully controllable synthetic ion channels. However, both insertion efficiency and stability of existing DNA-based membrane channels leave much room for improvement. Here, we demonstrate an approach to overcoming the unfavorable DNA-lipid interactions that hinder the formation of a stable transmembrane pore. Our all-atom MD simulations and experiments show that the insertion-driving cholesterol modifications can cause fraying of terminal base pairs of nicked DNA constructs, distorting them when embedded in a lipid bilayer. Importantly, we show that DNA nanostructures with no backbone discontinuities form more stable conductive pores and insert into membranes with a higher efficiency than the equivalent nicked constructs. Moreover, lack of nicks allows design and maintenance of membrane-spanning helices in a tilted orientation within the lipid bilayer. Thus, reducing the conformational degrees of freedom of the DNA nanostructures enables better control over their function as synthetic ion channels.

Ethidium bromide interactions with DNA: an exploration of a classic DNA-ligand complex with unbiased molecular dynamics simulations.

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)2 also known as the Drew–Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 μs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.

Synergy between Protein Positioning and DNA Elasticity: Energy Minimization of Protein-Decorated DNA Minicircles.

The binding of proteins onto DNA contributes to the shaping and packaging of the genome as well as to the expression of specific genetic messages. With a view to understanding the interplay between the presence of proteins and the deformation of DNA involved in such processes, we developed a new method to minimize the elastic energy of DNA fragments at the mesoscale level. Our method makes it possible to obtain the optimal pathways of protein-decorated DNA molecules for which the terminal base pairs are spatially constrained. We focus in this work on the deformations induced by selected architectural proteins on circular DNA. We report the energy landscapes of DNA minicircles subjected to different levels of torsional stress and containing one or two proteins as functions of the chain length and spacing between the proteins. Our results reveal cooperation between the elasticity of the double helix and the structural distortions of DNA induced by bound proteins. We find that the imposed mechanical stress influences the placement of proteins on DNA and that the proteins, in turn, modulate the mechanical stress and thereby broadcast their presence along DNA.

Potential Unwinding of Double-Stranded DNA upon Binding to a Carbon Nitride Polyaniline (C3N) Nanosheet.

Recently, carbon nitride polyaniline (C3N) had attracted considerable attention from many scientific fields after its successful synthesis. However, thus far, limited efforts were devoted to reveal its potential effect to biomolecules, which correlated intimately with its further utilization. In this study, by using a molecular dynamics (MD) simulation approach, we investigated in detail the interaction between C3N and a double-stranded DNA (dsDNA) segment to expose the underlying biological effect of C3N to dsDNA and the corresponding molecular basis. MD simulation results demonstrated that dsDNA presented serious damages upon adsorption onto a C3N nanosheet with the terminal base pairs denaturized, unwound, and directly packing on the C3N surface, which implied that C3N was potentially deleterious to biomolecules. This binding/unwinding process was mainly guided by a combination of van der Waals and π-π stacking interactions together with a continuous lateral migration of dsDNA. Moreover, the nanoscale dewetting also played an important role during the adsorption. These findings revealed the potential bio-effect of the C3N nanomaterial and its molecular mechanism, which might benefit the future applications of C3N-based nanostructures.

Salt dependent mesoscopic model for RNA at multiple strand concentrations

Mesoscopic models can be used for the description of the thermodynamic properties of RNA duplexes. With the use of experimental melting temperatures, its parametrization can provide important insights into its hydrogen bonds and stacking interactions as has been done for high sodium concentrations. However, the RNA parametrization for lower salt concentrations is still missing due to the limited amount of published melting temperature data. While the Peyrard-Bishop (PB) parametrization was found to be largely independent of strand concentrations, it requires that all temperatures are provided at the same strand concentrations. Here we adapted the PB model to handle multiple strand concentrations and in this way we were able to make use of an experimental set of temperatures to model the hydrogen bond and stacking interactions at low and intermediate sodium concentrations. For the parametrizations we make a distinction between terminal and internal base pairs, and the resulting potentials were qualitatively similar as we obtained previously for DNA. The main difference from DNA parameters, was the Morse potentials at low sodium concentrations for terminal r(AU) which is stronger than d(AT), suggesting higher hydrogen bond strength.

Ancillary Ligand in Ternary CuII Complexes Guides Binding Selectivity toward Minor-Groove DNA.

Copper-containing compounds known as Casiopeínas are biologically active molecules which show promising antineoplastic effects against several cancer types. Two possible hypotheses regarding the mode of action of the Casiopeínas have emerged from the experimental evidence: the generation of reactive oxygen species or the ability of the compounds to bind and interact with nucleic acids. Using robust molecular dynamics simulations, we investigate the interaction of four different Casiopeínas with the DNA duplex d(GCACGAACGAACGAACGC). The studied copper complexes contain either 4–7- or 5–6-substituted dimethyl phenanthroline as the primary ligand and either glycinate or acetylacetonate as the secondary ligand. For statistical significance and to reduce bias in the simulations, four molecules of each copper compound were manually placed at a distance of 10 Å away from the DNA and 20 independent molecular dynamics simulations were performed, each reaching at least 30 μs. This time scale allows us to reproduce expected DNA terminal base-pair fraying and also to observe intercalation/base-pair eversion events generated by the compounds interacting with DNA. The results reveal that the secondary ligand is the guide toward the mode of binding between the copper complex and DNA in which glycinate prefers minor-groove binding and acetylacetonate produces base-pair eversion and intercalation. The CuII complexes containing glycinate interact within the DNA minor groove which are stabilized principally by the hydrogen bonds formed between the amino group of the aminoacidate moiety, whereas the compounds with the acetylacetonate do not present a stable network of hydrogen bonds and the ligand interactions enhance DNA breathing dynamics that result in base-pair eversion.

Molecular dynamics simulations of acyclic analogs of nucleic acids for antisense inhibition

For antisense applications, oligonucleotides must be chemically modified to be resistant to endogenous nucleases. Until now, antisense oligonucleotide (ASO) analogs have been synthesized and then tested for their ability to duplex with a target nucleic acid, usually RNA. In this work, using molecular dynamics calculations simulations, we systematically tested a series of chemically modified analogs in which the 2-deoxyribose was substituted for by one or two methylene groups on each side of the phosphate backbone, producing four compounds, of which three were previously unknown. We used a 9-mer sequence of which the solution structure has been determined by NMR spectroscopy and tested the ability to form stable duplexes of these acyclic analogs to both DNA and RNA. In only one case out of eight, we unexpectedly found the formation of a stable duplex with complementary RNA. We also applied limitations on end fraying because of the terminal AT base pairs, in order to eliminate this as a factor in the comparative results. We consider this a predictive method to potentially identify target ASO analogs for synthesis and testing for antisense drug development.

DNA minor-groove binder Hoechst 33258 destabilizes base-pairing adjacent to its binding site

Understanding the dynamic interactions of ligands to DNA is important in DNA-based nanotechnologies. By structurally tracking the dissociation of Hoechst 33258-bound DNA (d(CGCAAATTTGCG)2) complex (H-DNA) with T-jump 2D-IR spectroscopy, the ligand is found to strongly disturb the stability of the three C:G base pairs adjacent to A:T the binding site, with the broken base pairs being more than triple at 100 ns. The strong stabilization effect of the ligand on DNA duplex makes this observation quite striking, which dramatically increases the melting temperature and dissociation time. MD simulations demonstrate an important role of hydration water and counter cations in maintaining the separation of terminal base pairs. The hydrogen bonds between the ligand and thymine carbonyls are crucial in stabilizing H-DNA, whose breaking signal appearing prior to the complete dissociation. Thermodynamic analysis informs us that H-DNA association is a concerted process, where H cooperates with DNA single strands in forming H-DNA.